IAP binding compounds

ABSTRACT

IAP binding molecules and compositions including these are disclosed. The IAP binding molecules interact with IAPs (inhibitor of apoptosis proteins) in cells and may be used to modify apoptosis in cells treated with such molecules. Embodiments of these compounds have a K d  of less than 0.1 micromolar. Methods of using these IAP binding molecules for therapeutic, diagnostic, and assay purposed are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/926,283, filed Jun. 25, 2013; which is a continuation of U.S.application Ser. No. 13/152,644, filed Jun. 3, 2011 (now abandoned);which is a continuation of U.S. application Ser. No. 12/248,494, filedOct. 9, 2008 (now U.S. Pat. No. 7,968,590); which is a divisional ofU.S. application Ser. No. 11/184,503, filed Jul. 15, 2005 (now U.S. Pat.No. 7,456,209); which claims priority to U.S. Provisional App. No.60/588,050, filed Jul. 15, 2004, all of which are incorporated byreference herein.

BACKGROUND

Apoptosis, programmed cell death, plays a central role in thedevelopment and homeostasis of all multi-cellular organisms. Alterationsin apoptotic pathways have been implicated in many types of humanpathologies, including developmental disorders, cancer, autoimmunediseases, as well as neuro-degenerative disorders.

Programmed cell death pathways have become targets for the developmentof therapeutic agents. In some cases because it is easier to destroydiseased cells rather than to sustain them, anti-cancer therapies usingpro-apoptotic agents such as conventional radiation and chemo-therapyhave been used to trigger activation of the mitochondria-mediatedapoptotic pathways. However, these therapies lack molecular specificity,and more specific molecular targets are needed.

Apoptosis is executed primarily by activated caspases, a family ofcysteine proteases with aspartate specificity in their substrates.Caspases are produced in cells as catalytically inactive zymogens andmust be proteolytically processed to become active proteases duringapoptosis. In normal surviving cells that have not received an apoptoticstimulus, most caspases remain inactive. Even if some caspases areaberrantly activated, their proteolytic activity can be fully inhibitedby a family of evolutionarily conserved proteins called IAPs (inhibitorsof apoptosis proteins) (Deveraux & Reed, Genes Dev. 13: 239-252, 1999).Each of the IAPs contains 1-3 copies of the so-called BM (baculoviralIAP repeat) domain and directly interacts with and inhibits theenzymatic activity of mature caspases. Several distinct mammalian IAPsincluding XIAP, survivin, and LIVIN/ML-IAP, (Kasof and, mes, J. Biol.Chem. 276: 3238-3246, 2001; Vuc/ic et al. Curr. Biol. 10: 1359-1366,2000; Ashhab et al. FEBS Lett. 495: 56-60, 2001), have been identifiedand they exhibit anti-apoptotic activity in cell culture (Deveraux &Reed, 1999, supra). As IAPs are expressed in most cancer cells, they maydirectly contribute to tumor progression and subsequent resistance todrug treatment.

In normal cells signaled to undergo apoptosis, however, the IAP-mediatedinhibitory effect must be removed, a process at least in part performedby a mitochondrial protein named Smac, second mitochondria-derivedactivator of caspases; (Du et al. Cell 102: 33-42,2000) or DIABLO(direct IAP binding protein with low pI; Verhagen et al. Cell 102:43-53,2000). Smac/DIABLO, synthesized in the cytoplasm, is targeted tothe inter-membrane space of mitochondria. Upon apoptotic stimuli, Smacis released from mitochondria back into the cytosol, together withcytochrome c. Whereas cytochrome c induces multimerization of Apaf-1 toactivate procaspase-9 and procaspase-3, Smac eliminates the inhibitoryeffect of multiple IAPs. Smac interacts with all IAPs that have beenexamined to date, including XIAP, c-IAP1, c-IAP2, ML-IAP, and survivin.Smac appears to be a regulator of apoptosis in mammals. In addition tothe inhibition of caspases, overexpressed IAPs can function to bind Smacand prevent it from binding to XIAP and releasing caspases (Vucic et.al., Biochem. J. 385(Pt 1):11-20, 2005).

Smac is synthesized as a precursor molecule of 239 amino acids; theN-terminal 55 residues serve as the mitochondria targeting sequence thatis removed after import. The mature form of Smac contains 184 aminoacids and behaves as an oligomer in solution. Smac and various fragmentsof it have been proposed for use as targets for identification oftherapeutic agents. The biological activity of Smac is believed to berelated to binding of its N-terminal four residues to a featured surfacegroove in a portion of XIAP referred to as the BIR3 domain. This bindingprevents XIAP from exerting its apoptosis-suppressing function in thecell. The N-terminal tetrapeptides from IAP binding proteins of theDrosophila pro-apoptotic proteins Hid, Grim and Reaper are believed tofunction in the same manner.

Commonly-owned co-pending International Application No. PCT/US02/17342,filed May 31, 2002 and incorporated herein by reference in its entirety,discloses assays for use in high throughput screening of agents thatbind to a BIR domain of an IAP, thereby relieving IAP-mediatedsuppression of apoptosis. The assays utilize a labeled IAP-bindingpeptide or peptidomimetic that binds to a BIR domain of an IAP, whereinat least one measurable feature of the label changes as a function ofthe IAP binding compound being bound to the IAP or free in solution. TheBIR domain of an IAP is contacted with the labeled IAP peptide orpeptidomimetic to form a complex, and the complex is exposed to acompound to be tested for BIR binding. Displacement of the labeled IAPpeptide or peptidomimetic from the complex, if any, by the testcompound, is measured.

Disadvantages in the use of peptides for in vivo administration asdiagnostic or therapeutic agents may include their short half-life dueto proteolytic degradation of the peptide in the body, low absorptionthrough intestinal walls, potential immunogenic reactions, as well asexpense involved in peptide synthesis. It would be beneficial to preparenon-peptidic IAP binding compounds that have comparable biologicalactivity of bioactive peptides, but possess improved pharmacologicalproperties and are easier or less expensive to synthesize.

In connection with the Smac tetrapeptides it would be a significantadvance in the art to develop IAP-binding compounds which may be used topromote apoptosis, while also having the improved properties associatedwith non-peptide compounds. Such compounds can be used as diagnostic andtherapeutic agents in the treatment of apoptosis related conditions.

SUMMARY OF THE INVENTION

An embodiment of the present invention is a compound, or compositioncomprising a compound, of the general formula (2):

wherein: A₁ and A₂ are independently hydrogen, alkyl, aryl, or alkylarylgroup, R_(1a) is H or a methyl group; R_(1b) is an alkyl or aryl group;X₁ is —O—, —S—, —CH₂—, or —NH— group, and J is —CH—, or —N— group,provided that when J is —N—, X₁ is —CH₂—, or —NH— group; Y is H, or analkyl group; Z is —OH, aryloxy, alkoxy, benzyloxy, benzyloxy, amino,arylamino, alkylamino, benzylamino group; R₂ is a detectable label oris:

M is alkylene, alkenylene, alkynlene, heteroalkylene, heteroalkenylene,or heteroalkynlene group, G is selected from a bond, —O—; —N(R_(2d))—where R_(2d) is H, alkyl, cycloalkyl, or aryl; or —S(O)_(m)— where m is0, 1, or 2; and R₁₀ is cycloalkyl, aryl, heterocycloalkyl,heterocycloalkenyl, or heteroaryl; n is independently the integer 0, 1,2, 3, 4, or 5.

Another embodiment of the present invention is a compound, orcomposition including a compound, of the general formula (3):

where A₁ is H, lower alkyl, or optionally-substituted lower alkyl group;R_(1a) and R_(1b) are separately H, lower alkyl, optionally-substitutedlower alkyl, lower alkylene, optionally substituted lower alkylenegroup; or A₁ together with either R_(1a) or R_(1b) form an optionallysubstituted heterocycloalkyl group of 3 to 6 atoms; Y is H, an alkylgroup, an alkynyl group, a cycloalkyl group of 3 to 7 carbon atoms,aryl, heteroaryl, arylalkyl, optionally-substituted versions of thesegroups, hydroxy substituted versions of these groups, or Y together withZ, M, G, or R₁₀ forms a carbocyclic ring, or a heterocyclic ringcontaining 1 to 5 heteroatoms, where Y is linked to Z, M, G, or R₁₀; Zis H, alkyl, hydroxy, amino, alkylamino, diakylamino, alkoxy,cycloalkyl, cycloalkyloxy, aryl, heteroaryl, aryloxy, or heteroaryloxygroup; or Z together with Y, M, G, or R₁₀ form a carbocyclic ring, or aheterocyclic ring containing 1 to 5 heteroatoms, where Z is linked to Y,M, G, or R₁₀; M is an optionally-substituted alkyl, alkenyl, or alkynylgroup; an optionally-substituted alkyl, alkenyl, or alkynyl group of 1to 5 carbon atoms; an optionally-substituted alkylene, alkenylene, oralkynylene group; or an optionally-substituted alkylene, alkenylene, oralkynylene group of 1 to 5 carbon atoms; G is a bond, a heteroatom,—(C═O)—; —S(O)_(t)— where t=0, 1, or 2; —NR₁₈—; —NCOR₁₈—; or—NS(O)_(x)R₁₈— where x=0, 1, or 2, and R₁₈ is lower alkyl,optionally-substituted lower alkyl, or cycloalkyl or R₁₈ is containedwithin a carbocyclic, or heterocyclic ring containing 1 to 5heteroatoms, where R₁₈ is linked to Z, M, or R₁₀; R₁₀ is an aryl, aheteroaryl group, a fused aryl, a fused heteroaryl group; or R₁₀ is anyone of structures (4a), (4b), (4c) or (4d):

where X₂ is a heteroatom and independently groups R₁₁, R′₁₁, R₁₂, any ofR₁₃₋₁₇, or any of R₁₄₋₁₇ is H, halogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, hydroxyl, alkoxy, polyalkylether, amino, alkylamino,dialkylamino, alkyloxyalkyl, sulfonate, aryloxy or heteroaryloxy;independently R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ is H,optionally-substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,hydroxyl, alkoxy, polyalkylether, amino, alkylamino, dialkylamino,alkyloxyalkyl, aryloxy, or heteroaryloxy; or independently R₁₁, R′₁₁,R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ is acyl or acetyl groups,carboxylate, sulfonate, sulfone, imine, or oxime groups; or groups R₁₁,R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ is contained within acarbocyclic ring, or a heterocyclic ring containing 1 to 5 heteroatoms,and linked to groups at position Y, Z, M, G, R_(1l), R′₁₁, R₁₂, any ofR₁₃₋₁₇, or any of R₁₄₋₁₇.

Another embodiment is compound, or a composition comprising a compound,of the general formula (5)

where A₁ is H, or lower alkyl; R_(1a) is H; R_(1b) is lower alkyl group;Y is an alkyl group, a cycloalkyl group of 3 to 7 carbon atoms,optionally substituted versions of these groups, hydroxy substitutedversions of these groups; Z_(1a) and Z_(1b) are independently an H,hydroxy, alkoxy, aryloxy, or heteroaryloxy group; M is anoptionally-substituted alkyl or an optionally-substituted alkylene groupof 1 to 5 carbon atoms; G is a bond, a heteroatom, or —NCOR₁₈— and R₁₈is lower alkyl, optionally-substituted lower alkyl group; R₁₀ is anyoneof structures (4a), (4b), (4c) or (4d):

where X₂ is a heteroatom and independently groups R₁₁, R′₁₁, R₁₂, any ofR₁₃₋₁₇, or any of R₁₄₋₁₇ are H, halogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, hydroxyl, alkoxy, polyalkylether, amino, alkylamino,dialkylamino, alkyloxyalkyl, sulfonate, aryloxy or heteroaryloxy;independently R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ are H,optionally-substituted alkyl, aryl, alkenyl, alkynyl, heteroaryl,hydroxyl, alkoxy, polyalkylether, amino, alkylamino, dialkylamino,alkyloxyalkyl, aryloxy, or heteroaryloxy; independently R₁₁, R′₁₁, R₁₂,any of R₁₃₋₁₇, or any of R₁₄₋₁₇ are acyl or acetyl groups, carboxylate,sulfonate, sulfone, imine, or oxime groups; or groups R₁₁, R′₁₁, R₁₂,any of R₁₃₋₁₇, or any of R₁₄₋₁₇ are contained within a carbocyclic ring,or a heterocyclic ring containing 1 to 5 heteroatoms, and linked togroups at position Y, Z_(1a), Z_(1b), M, G, R₁₁, R′₁₁, R₁₂, any ofR₁₃₋₁₇₃ or any of R₁₄₋₁₇.

In a preferred embodiment the present invention is compound, or acomposition comprising a compound, of the general formula (5)

where A₁ is H, or lower alkyl; R_(1a) is H; R_(1b) is lower alkyl group;Y is an alkyl group, a cycloalkyl group of 3 to 7 carbon atoms,optionally substituted versions of these groups, hydroxy substitutedversions of these groups; Z_(1a) and Z_(1b) are independently an H,hydroxy, alkoxy, aryloxy, or heteroaryloxy group; M is anoptionally-substituted alkyl or an optionally-substituted alkylene groupof 1 to 5 carbon atoms; G is a bond, a heteroatom, or —NCOR₁₈— and R₁₈is lower alkyl, optionally-substituted lower alkyl group; R₁₀ is anyoneof structures (4a), (4b), (4c) or (4d):

where X₂ is a heteroatom and independently groups R₁₁, R′₁₁, R₁₂, any ofR₁₃₋₁₇, or any of R₁₄₋₁₇ are H, halogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, hydroxyl, alkoxy, polyalkylether, amino, alkylamino,dialkylamino, alkyloxyalkyl, sulfonate, aryloxy or heteroaryloxy;independently R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ are H,optionally-substituted alkyl, aryl, alkenyl, alkynyl, heteroaryl,hydroxyl, alkoxy, polyalkylether, amino, alkylamino, dialkylamino,alkyloxyalkyl, aryloxy, or heteroaryloxy; independently R₁₁, R′₁₁, R₁₂,any of R₁₃₋₁₇, or any of R₁₄₋₁₇ are acyl or acetyl groups, carboxylate,sulfonate, sulfone, imine, or oxime groups; or groups R₁₁, R′₁₁, R₁₂,any of R₁₃₋₁₇, or any of R₁₄₋₁₇ are contained within a carbocyclic ring,or a heterocyclic ring containing 1 to 5 heteroatoms, and linked togroups at position Y, Z_(1a), Z_(1b), M, G, R₁₁, R′₁₁, R₁₂, any ofR₁₃₋₁₇, or any of R₁₄₋₁₇. Even more preferably, X₂ is nitrogin.

Further embodiments of the present invention include molecules andcompositions that may be useful to modify or regulate apoptosis incells. These IAP binding molecules can bind to a variety of IAP's(Inhibitor of Apoptosis Proteins). These molecules may be monomers ordimers and may also include a detectable label or therapeutic moiety andcan be formulated as pharmaceutical or diagnostic compositionscontaining these molecules. Methods for using these compounds astherapeutic and diagnostic agents are also described.

The IAP binding molecules of the present invention, which can also bereferred to as IAP binding cargo molecules, can permeate, betransfected, or otherwise be actively or passively transported intocells and can be used to displace IAPs from other proteins like caspasesor Smac in cells. At least a portion of the IAP binding-cargo moleculebinds to a BIR domain of an IAP. The IAP binding cargo molecule mayprovide a therapeutic effect for a cell proliferation disorder and caninclude additional therapeutic, diagnostic, or other substituents in themolecule. Embodiments of the IAP binding molecules include derivativesof pyrrolidine that bind to a BIR domain of an IAP.

Embodiments of the present invention include IAP binding cargo moleculesand pharmaceutically acceptable salts thereof having the generalstructure of formula (2):

wherein: A₁ and A₂ can independently be hydrogen, alkyl, aryl, oralkylaryl group, R_(1a) can be H or a methyl group; R_(1b) may be analkyl or aryl group, in some embodiments R_(1b) is methyl, ethyl,n-propyl, isopropyl, or ethenyl group; X₁ can be —O—, —S—, —CH₂—, or—NH— group, and J can be —CH—, or —N— group, provided that when J is—N—, X₁ is —CH₂—, or an —NH— group; Y can be H, or an alkyl group; Z canbe H, —OH, aryloxy, alkoxy, benzyloxy, amino, arylamino, alkylamino,benzylamino group, in some embodiments Z is —OH, aryloxy, alkoxy,benzyloxy, benzyloxy, amino, arylamino, alkylamino, benzylamino group;R₂ can include a detectable label or can be:

where R_(2a) can be an aryl, cycloalkyl, optionally substituted aralkyl,or cycloalkylalkyl group; R_(2b) can be H or alkyl group, R_(2c) can bearyl, cycloalkyl, optionally substituted aralkyl, or cycloalkylalkyl,heterocycloalkyl, heterocycloalkenyl, heteroaryl, or cycloalkylarylgroup. In some embodiments R_(2c) is tetrahydronaphthyl or substitutedtetrahydronapthyl group, most preferably R_(2c) is

Chiral carbons (i*) for (i*=3 to 8) may independently have an (R) or (S)configuration; M can be alkylene, alkenylene, alkynlene, heteroalkylene,heteroalkenylene, heteroalkynlene group, in some embodiments M is:

In some embodiments G can be selected from a bond (i.e., G is absent),—O—; —N(R_(2d))— where R_(2d) can be H, alkyl, cycloalkyl, or aryl; or—S(O)_(m)— where m is 0, 1, or 2;

R₁₀ can be cycloalkyl, aryl, heterocycloalkyl, heterocycloalkenyl, orheteroaryl; in some embodiments R₁₀ is:

where R₃, R′₃, R₄, R₅, R′₅, R₆, R₇, R₈ and R₉ can each independently H,methyl, ethyl, n-propyl, isopropyl, halo, cyano, —(CH₂)_(p)C(═O)OH,—(CH₂)_(p)C(═O)O-alkyl, —(CH₂)_(p)C(═O)NH₂; n and p are integers andpreferably n is independently the integer 0, 1, 2, 3, 4, or 5 and p isindependently the integer 0, 1, 2, or 3; preferably at least one R₃,R′₃, R₄, and R′₅, R₅ or at least two of R₆, R₇, R₈ and R₉ are eachindependently H, methyl, ethyl, n-propyl, isopropyl, halo, or cyano;provided that when one or more of R₃, R′₃, R′₅, and R₅ is isopropyl, R₄is other than isopropyl; provided that when R₄ is isopropyl, R₃, R′₃,R′₅, and R₅ are each independently other than isopropyl; provided thatwhen R₈ is isopropyl, R₉ is other than isopropyl; and provided that in atherapeutic composition, (2) is not the structure where R₂ is

and R_(2c) is

andwhere A₁ is H, A₂ is methyl, R_(1a) is H, R_(1b) is methyl, X₁ is —NH—,J is —CH—, Y is t-butyl, Z is (—OC₆H₅) and (3*) has an (S)configuration, (4*) has an (S) configuration, (5*) has an (5) or (R)configuration, (6*) has an (S) or (R) configuration, and (7*) has an (R)configuration. Some embodiments of compounds of structure (2) have aK_(d) as determined by the methods described, for example, in Example 1of less than 100 micromolar, preferably less than 1 micromolar, and evenmore preferably less than 0.1 micromolar.

Some embodiments of the IAP binding compounds or IAP binding cargomolecules of structure (2), where A₂ is H, X₁ is —NH—, J is —CH—, and nis 0 for R₂, can be depicted by structure (3):

In some embodiments of compounds of structure (3), A₁ can be H, loweralkyl, or optionally-substituted lower alkyl group; R_(1a) and R_(1b)can separately be H, lower alkyl, optionally substituted lower alkyl,lower alkylene, optionally substituted lower alkylene group; or A₁together with either R_(1a) or R_(1b) can form an optionally substitutedheterocycloalkyl group of 3 to 6 atoms;

Y can be H, an alkyl group, an alkyl group of 1 to 10 carbon atoms, abranched alkyl group of 1 to 10 carbon atoms, an alkynyl group, acycloalkyl group of 3 to 7 carbon atoms, aryl, heteroalkynyl,heteroaryl, or arylalkyl group; optionally-substituted versions of theaforementioned groups; hydroxy substituted versions of theaforementioned groups; or Y together with Z, M, G, or R₁₀ forms anoptionally substituted carbocyclic ring, or an optionally substitutedheterocyclic ring containing 1 to 5 heteroatoms, where Y is linked to Z,M, G, or R₁₀; preferably Y is linked to M, G, or R₁₀ by any number ofatoms up to about 20 atoms.

Z can be H, alkyl, hydroxy, amino, alkylamino, dialkylamino, alkoxy,cycloalkyl, cycloalkyloxy, aryl, heteroaryl, aryloxy, or heteroaryloxygroup; or Z together with Y, M, G, or R₁₀ form an optionally substitutedcarbocyclic ring, or an optionally substituted heterocyclic ringcontaining 1 to 5 heteroatoms, where Z is linked to Y, M, G, or R₁₀;preferably Z is linked to Y, M, G, or R₁₀ by any number of atoms up toabout 20 atoms.

M can be an optionally substituted alkyl, alkenyl, or alkynyl group; anoptionally substituted alkyl, alkenyl, or alkynyl group of 1 to 5 carbonatoms; an optionally substituted alkylene, an alkenylene, or alkynylenegroup; or an optionally substituted alkylene, alkenylene, or alkynylenegroup of 1 to 5 carbon atoms.

G can be absent (a bond), or a heteroatom including —O—; —NH—; —(C═O)—;—S(O)_(t)— where t is the integer 0, 1, or 2; —NR₁₈—; —NCOR₁₈—; or—NS(O)_(x)R₁₈— where x is the integer 0, 1, or 2, and R₁₈ can be loweralkyl, optionally-substituted lower alkyl, or cycloalkyl or R₁₈ iscontained within an optionally substituted carbocyclic, or optionallysubstituted heterocyclic ring containing 1 to 5 heteroatoms, where R₁₈is linked to Z, M, or R₁₀, preferably R₁₈ is linked to Z, M, or R₁₀, byany number of atoms up to about 20 atoms.

R₁₀ can be an aryl, a heteroaryl group, a fused aryl, a fused heteroarylgroup or optionally substituted versions of these groups; or R₁₀ can beany one of structures (4a), (4b), (4c), or (4d):

where X₂ is a heteroatom in structures (4a) or (4b), or X₂ is acarbon-carbon bond as illustrated in structures (4c) or (4d), andindependently groups R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ canbe H, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, hydroxyl,alkoxy, polyalkylether, amino, alkylamino, dialkylamino, alkyloxyalkyl,aryloxy or heteroaryloxy; or independently R₁₁, R′₁₁, R₁₂, any ofR₁₃₋₁₇, or any of R₁₄₋₁₇ can be H, optionally-substituted alkyl,alkenyl, alkynyl, aryl, heteroaryl, hydroxyl, alkoxy, polyalkylether,amino, alkylamino, dialkylamino, alkyloxyalkyl, aryloxy, orheteroaryloxy; or independently R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any ofR₁₄₋₁₇ can be acyl or acetyl groups, carboxylate, sulfonate, sulfone,imine, or oxime groups; or groups R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or anyof R₁₄₋₁₇ can be contained within an optionally substituted carbocyclicring, or an optionally substituted heterocyclic ring containing 1 to 5heteroatoms, and can be linked to groups at position Y, Z, M, G, R₁₁,R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇, preferably these groups arelinked by any number of atoms up to about 20 atoms. Some embodiments ofcompounds of structure (3) have a K_(d) as determined by the methodsdescribed, for example, in Example 1 of less than 100 micromolar,preferably less than 1 micromolar, and even more preferably less than0.1 micromolar.

Some embodiments include compounds of structure (5) where:

A₁ can be H, or lower alkyl, or A₁ and R_(1b) together form a ring of3-5 atoms;

R_(1a) can be H; R_(1b) can be a lower alkyl group, or together with A₁forms a ring of 3 to 5 atoms;

Y can be an alkyl group, an alkyl group of 1 to 10 carbon atoms, abranched alkyl group of 1 to 10 carbon atoms, an alkynyl group,heteroalkynyl, a cycloalkyl group of 3 to 7 carbon atoms, optionallysubstituted versions of the aforementioned groups, hydroxy substitutedversions of the aforementioned groups, or Y together with Z_(1a),Z_(1b), or R₁₀ forms an optionally substituted carbocyclic ring, or anoptionally substituted heterocyclic ring containing 1 to 5 heteroatoms,where Y can be linked to Z_(1a), Z_(1b), or R₁₀; preferably Y is linkedto Z_(1a), Z_(1b), or R₁₀ by any number of atoms up to about 20 atoms.

Z_(1a) and Z_(1b) can independently be an H, hydroxy, amino, alkylamino,dialkylamino, alkoxy, aryloxy, or heteroaryloxy group; or Z_(1a),Z_(1b), together with Y or R₁₀ form a carbocyclic ring, or aheterocyclic ring containing 1 to 5 heteroatoms, where Z_(1a) or Z_(1b),is linked to Y or R₁₀; preferably Z_(1a) or Z_(1b), is linked to Y orR₁₀ by any number of atoms up to about 20 atoms.

M can be an optionally-substituted alkyl or an optionally-substitutedalkylene group of 1 to 5 carbon atoms.

G can be absent (a bond), or a heteroatom including —O—; —NH—; —(C═O)—;—NR₁₈—; —NCOR₁₈—; or —NS(O)_(x)R₁₈— where x=0, 1, or 2, and R₁₈ can belower alkyl, optionally-substituted lower alkyl group.

R₁₀ can be aryl, a heteroaryl group, or R₁₀ can be anyone of structures(4a), (4b), (4c), or (4d):

where X₂ can be a heteroatom in structures (4a) or 4(b) or X₂ is acarbon-carbon bond as illustrated in structures (4c) or (4d), andindependently groups R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ canbe H, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, hydroxyl,alkoxy, polyalkylether, amino, alkylamino, dialkylamino, alkyloxyalkyl,sulfonate, aryloxy or heteroaryloxy; or independently R₁₁, R′₁₁, R₁₂,any of R₁₃₋₁₇, or any of R₁₄₋₁₇ can be H, optionally-substituted alkyl,alkenyl, alkynyl, aryl, heteroaryl, hydroxyl, alkoxy, polyalkylether,amino, alkylamino, dialkylamino, alkyloxyalkyl, aryloxy, orheteroaryloxy; or independently R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any ofR₁₄₋₁₇ can be acyl or acetyl groups, carboxylate, sulfonate, sulfone,imine, or oxime groups; or groups R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or anyof R₁₄₋₁₇ can be contained within a carbocyclic ring, or a heterocyclicring containing 1 to 5 heteroatoms, and linked to groups at position Y,Z_(1a), Z_(1b), M, G, R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇,preferably these groups are linked by any number of atoms up to about 20atoms. Some embodiments of compounds of structure (5) have a K_(d) asdetermined by the methods described, for example, in Example 1 of lessthan 100 micromolar, preferably less than 1 micromolar, and even morepreferably less than 0.1 micromolar.

Some embodiments include compounds of structure (5) where:

A₁ can be H, methyl, ethyl, or A₁ and R_(1b) together form a ring of 3-5atoms.

R_(1a) can be H; R_(1b) can be a methyl or ethyl group, or together withA₁ forms a ring of 3 to 5 atoms.

Y can be an alkyl group, an alkyl group of 1 to 10 carbon atoms, abranched alkyl group of 1 to 10 carbon atoms, an alkynyl group,heteroalkynyl, a cycloalkyl group of 3 to 7 carbon atoms, optionallysubstituted versions of the aforementioned groups; hydroxy substitutedversions of the aforementioned groups; or Y together with Z_(1a),Z_(1b), or R₁₀ forms a carbocyclic ring, or a heterocyclic ringcontaining 1 to 5 heteroatoms, where Y is linked to Z_(1a), Z_(1b), orR₁₀; preferably Y is linked to Z_(1a), Z_(1b), or R₁₀ by any number ofatoms up to about 20 atoms.

Z_(1a) and Z_(1b) can independently be an H, hydroxy, amino, alkylamino,diakylamino, alkoxy, aryloxy, or heteroaryloxy group; or Z_(1a), Z_(1b),together with Y or R₁₀ form a carbocyclic ring, or a heterocyclic ringcontaining 1 to 5 heteroatoms, where Z_(1a) or Z_(1b), is linked to Y orR₁₀; preferably Z_(1a) or Z_(1b), is linked to Y or R₁₀ by any number ofatoms up to about 20 atoms.

M can be an optionally-substituted alkyl or an optionally-substitutedalkylene group of 1 to 5 carbon atoms.

G can be absent (a bond), or a heteroatom including —O—; —NH—; —(C═O)—;—NR₁₈—; —NCOR₁₈—; or —NS(O)_(x)R₁₈— where x can be the integer 0, 1, or2, and R₁₈ can be lower alkyl, optionally-substituted lower alkyl group.

R₁₀ can be a fused aryl, a fused heteroaryl group, or preferably R₁₀ isanyone of structures (4a), (4b), (4c), or (4d):

where X₂ can be a heteroatom (4a) or (4b) or X₂ can be a carbon-carbonbond (4c) or (4d), and independently groups R₁₁, R′₁₁, R₁₂, any ofR₁₃₋₁₇, or any of R₁₄₋₁₇ can be H, halogen, alkyl, alkenyl, alkynyl,aryl, heteroaryl, hydroxyl, alkoxy, polyalkylether, amino, alkylamino,dialkylamino, alkyloxyalkyl, sulfonate, aryloxy or heteroaryloxy; orindependently R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ can be H,optionally-substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,hydroxyl, alkoxy, polyalkylether, amino, alkylamino, dialkylamino,alkyloxyalkyl, aryloxy, or heteroaryloxy; or independently R₁₁, R′₁₁,R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ can be acyl or acetyl groups,carboxylate, sulfonate, sulfone, imine, or oxime groups; or groups R₁₁,R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ can be contained within acarbocyclic ring, or a heterocyclic ring containing 1 to 5 heteroatoms,and linked to groups at position Y, Z_(1a), Z_(1b), M, G, R₁₁, R′₁₁,R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇, preferably these groups are linkedby any number of atoms up to about 20 atoms.

Some embodiments include compounds of structure (5) where:

where A₁ can be H, or a methyl group; R_(1a) is H; R_(1b) can be amethyl or ethyl group.

In structure (5) Y can be an alkyl group, an alkyl group of 1 to 10carbon atoms, a branched alkyl group of 1 to 10 carbon atoms, an alkynylgroup, heteroalkynyl, a cycloalkyl group of 3 to 7 carbon atoms,optionally substituted versions of the aforementioned groups, hydroxysubstituted versions of the aforementioned groups, or Y together withR₁₀ forms a carbocyclic ring, or a heterocyclic ring containing 1 to 5heteroatoms, where Y is linked to R₁₀; preferably Y is linked to R₁₀ byany number of atoms up to about 20 atoms.

Z_(1a) and Z_(1b) can independently be an H, hydroxy, alkoxy, or aryloxygroup.

M can be methylene, an optionally-substituted alkyl or anoptionally-substituted alkylene group of 1 to 5 carbon atoms.

G can be absent (a bond), or a heteroatom including —O—; or —NH—,

R₁₀ can be an aryl, a heteroaryl group, or in some embodiments R₁₀ canbe a structure of formula (4a):

where X₂ is a heteroatom and independently groups R₁₁, R₁₂, or any ofR₁₄₋₁₇ can be H, or optional substituents including halogen, alkyl,aryl, alkenyl, alkynyl, heteroaryl, hydroxyl, alkoxy, polyalkylether,amino, alkylamino, dialkylamino, alkyloxyalkyl, sulfonate, aryloxy orheteroaryloxy; or independently R₁₁, R₁₂, or any of R₁₄₋₁₇ can be H,optionally-substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,hydroxyl, alkoxy, polyalkylether, amino, alkylamino, dialkylamino,alkyloxyalkyl, aryloxy, or heteroaryloxy; or independently R₁₁, R₁₂, orany of R₁₄₋₁₇ can be acyl or acetyl groups, carboxylate, sulfonate,sulfone, imine, or oxime groups; or groups R₁₁, R₁₂, or any of R₁₄₋₁₇can be contained within a carbocyclic ring, or a heterocyclic ringcontaining 1 to 5 heteroatoms, and linked to groups at position Y,Z_(1a), Z_(1b), M, G, R₁₁, R₁₂, or any of R₁₄₋₁₇, preferably thesegroups are linked by any number of atoms up to about 20 atoms.

IAP binding compounds or IAP binding cargo molecules in variousembodiments of formula (2), (3), or (5) may be used in the manufactureof a medicament for the therapeutic and/or prophylactic treatment of acancer or cellular proliferation condition (including developmentaldisorders, cancer, autoimmune diseases, as well as neuro-degenerativedisorders). The IAP binding compounds or IAP binding cargo molecules invarious embodiments of formula (2), (3), or (5) can be used in thepreparation of a drug for treating cancer or a cellular proliferationdisorder condition in a ready to use form. The drug can be administeredto a patient for treating or preventing cancer or a cellularproliferation disorder. In ready to use form refers to the compoundsbeing presentable for sale and may include the compounds in a tablet,liquid, or other form for administration, suitable packaging,instructions, and other items.

One embodiment of the invention is a method of treating cells or tissuethat can include administering to cells having a proliferation disorder,for example HeLa cells known to overexpress IAP (other cells may includebut are not limited to those with developmental disorders, cancer,autoimmune diseases, as well as neuro-degenerative disorders), an amountof the IAP binding compounds or IAP binding cargo molecules in variousembodiments of formula (2), (3), or (5) that is effective to reduce oreliminate the cellular proliferation disorder in the sample of cells ortissue.

A further embodiment of the present invention is a method of treatingdisorders associated with cell proliferation, including, but not limitedto proliferative disorders and diseases. Such methods includeadministration of the compounds of the present invention alone or incombination with other active agents, including pharmaceuticals andchemotherapeutic agents. For example, the dimmers of IAP bindingcompounds of the present invention may be administered alone or incombination with chemotherapeutic agents as is disclosed in commonlyowned U.S. Provisional Application No. 60/692,111, which is incorporatedherein by reference in its entirety.

The foregoing IAP binding compounds, as well as pharmaceuticallyacceptable salts and solvates thereof, may be formulated aspharmaceutical compositions or as diagnostic agents, or both. Thesepharmaceutical compositions and diagnostic agents may be used fortreatment and detection of cell proliferative disorders, as well as inscreening assays for the discovery and development of additionaldiagnostic and therapeutic agents for modifying cell proliferation anddetecting cell proliferative disorders.

The present invention includes an assay for use in high throughputscreening or rational drug design of IAP binding compounds that can,like the Smac tetrapeptide or its homologs in other species, bind to aBIR domain of an IAP thereby modifying, and preferably relievingIAP-mediated suppression of apoptosis. The binding of test compounds canbe used in the design of IAP binding compounds and IAP binding cargomolecules for the identification, prevention, and treatment of diseasesrelated to cell proliferation. The IAP binding cargo molecule orcompounds in embodiments of the present invention can bind to proteinssuch as through the BIR domain of an IAP. In some embodiments, the IAPbinding molecule interacts with the BIR3 domain of the protein XIAP orBIR2 domain of DIAP1. The IAP binding molecule can interact with theprotein through a specific binding groove of the BIR domain.

The assay includes the steps of providing a labeled IAP binding compoundor an IAP binding-cargo molecule of structure (2), (3), or (5), thatbinds to the appropriate BIR domain of the IAP, wherein preferably atleast one measurable feature of the labeled IAP binding compound changesas a function of the labeled IAP binding compound being bound to the IAPor free in solution. The assay may further include contacting the BIRdomain of an IAP with the labeled IAP binding compound under conditionsenabling binding of the labeled IAP binding compound with the BIRdomain, thereby forming a labeled BIR-bound IAP binding compound complexhaving the measurable feature. The labeled BIR-bound IAP bindingcompound complex may be contacted with other peptides, IAP bindingcompounds, or test compounds being developed, to measure the binding ofthe peptides, IAP binding compounds, or test compound for the BIR domainby measuring the displacement of the labeled IAP binding compound fromthe labeled BIR-bound IAP binding compound complex. Displacement of thelabeled IAP binding compound from the labeled BIR-bound IAP bindingcompound complex by the peptides, IAP binding compounds, or testcompound can be determined by measuring the change in the measurablefeature of the labeled IAP binding compound, thereby determining if thetest compound is capable of binding to the BIR domain of the IAP and thestrength of the interaction.

The present invention relates to the treatment of cell proliferationconditions and diseases and more specifically conditions where theactivity of IAP in cells, tissues or an individual is abnormal. Theinvention features molecules that are IAP binding compounds of structure(2), (3), or (5), that bind to IAPs such as but not limited to XIAP,c-IAP1, c-IAP2, ML-IAP and survivin in cells. The mimetic moleculesoptionally include an integral or linked cargo portion that can includea therapeutic or diagnostic functionality. The IAP binding compoundmolecules may be administered to cells, a tissue, or a patient in needof treatment or detection for a cell proliferation condition or disease.The need for treatment can be identified by contacting cells or atissue, preferably from the patient, with an IAP binding molecule havinga detectable label or cargo that changes when the molecule binds to anIAP in the tissue or cells. The binding of the IAP binding compoundmolecule with the IAP in the cells can be used to modify a cellproliferation condition or disease or it may be combined with othertherapeutic treatments such as radiation therapy. The activity of IAP inthe cells or the progress of a course of treatment for a cellproliferation condition or disease may be measured with an IAP bindingcargo molecule having a detectable label.

According to one aspect of the invention, a method of selectivelyidentifying neoplastic or cancer cells in a mixed population of cells isprovided. The method includes contacting the mixed cell population witha cell permeable IAP-binding cargo molecule of structure (2), (3), or(5), under conditions enabling the IAP-binding cargo molecule to bind aprotein like an IAP within the neoplastic cells, thereby selectivelyidentifying the neoplastic cells by a detectable property of the IAPbinding cargo molecule, and in some embodiments a change in a detectableproperty of the IAP binding cargo molecule upon complexation with IAP inthe neoplastic cells. The cells may be cultured cells or primary cellsfrom a patient (human or animal). Alternatively, the cells may bepresent within the patient, and the contacting accomplished byintroducing the IAP-binding cargo molecule into the patient.

In an embodiment of the IAP-binding cargo molecule, the cargo portion ofthe molecule comprises a dye label. In other embodiments, the cargoportion of the molecule can be, but is not limited to, an NMR-activenucleus or an MRI contrast agent. The selective identification oftissues or cells having IAP is performed through nuclear magneticresonance or magnetic resonance imaging. Alternatively, the labeledIAP-binding cargo molecule comprises a radioisotope and the selectiveidentification is performed through positron emission tomography.

Another aspect of the invention features a method of selectivelydamaging or inducing apoptosis in neoplastic cells by killing some orall of the neoplastic cells in a mixed population of cells. The methodincludes contacting a sample of the mixed cell population with anIAP-binding cargo molecule of formula (2), or (3), or (5). The IAPbinding portion of the molecule or the cargo portion of the molecule caninclude a moiety or substituent that is directly or indirectly toxic tocells such as but not limited to a radioisotope or a photosensitizingagent. The IAP binding portion of the molecule binds to a protein likean IAP within the neoplastic cells, where the toxic moiety of theIAP-binding cargo molecule directly or indirectly exerts its toxiceffect, thereby damaging or killing at least a portion the neoplasticcells in a mixed population of cells.

Another embodiment of the present invention is a composition thatincludes cells and an IAP-binding cargo molecule. The IAP binding cargomolecule binds to an IAP protein, such as XIAP, c-IAP1, c-IAP2, ML-IAP,or survivin, preferably it binds to a BIR surface groove of an IAPprotein. In some embodiments the IAP binding molecule binds to the BIR3surface groove of an XIAP protein. The IAP binding cargo molecule ofstructure (2), (3), or (5), can permeate or be transfected into thecells and can, for example, be used to displace one or more IAP proteinsfrom caspases in the cells or displace a protein like Smac sequesteredby IAPs. The IAP binding cargo molecule can have a detectable propertywhich is modified upon chemical, physical, or a combination of theseinteractions, of the IAP binding molecule with the IAP protein in thecells. This composition is useful as a control for monitoring thepresence of IAP in the cells undergoing treatment or for use as astandard in the detection of abnormal IAP levels in a sample of cells.The detectable property may be the emission of light by the cargoportion of the molecule which changes when the IAP bonding portion ofthe molecule binds to an IAP protein.

The IAP binding compounds or IAP binding cargo molecules of structure(2), (3), or (5) in embodiments of the invention may be characterized byhaving an IAP binding constant K_(d). In some embodiments K_(d) is lessthan about 10 μM, in other embodiments K_(d) is less than about 1 μM,and in still other embodiments K_(d) is less than about 0.1 μM asdetermined by the methods described in Example 1 or equivalents of thesemethods known to those skilled in the art. Molecules of formula (2),(3), or (5) having a K_(d) of less than about 10 μM can be used in an invitro binding assay with the BIR domain of an IAP and in someembodiments the BIR3 domain of XIAP. The IAP binding cargo molecule, andpreferably the cargo portion of the molecule includes but is not limitedto a fluorogenic group, a radioisotope, or other chromogenic group. Infurther embodiments the IAP binding cargo molecule may include anotherpeptide or peptidomimetic unit (e.g., dimer). The IAP binding cargomolecule may include an NMR-active nucleus or an MRI contrast agent andthe selective identification of the cargo portion of the moleculeperformed through nuclear magnetic resonance or magnetic resonanceimaging. The one or more cells in the composition may include but arenot limited to cells from a bodily fluid, tissue, tumor, fibroid,neoplastic cells, stem cells, nervous system cells or any combination ofthese from an animal, a mammal, or a human. The cells in the compositionmay be taken from tissue suspected of exhibiting an abnormal level ofIAP based upon physical examination, motor skill tests, or detection bypalpation of a lump in a part of the body. The composition may includeone or more pharmaceutically acceptable excipients.

Another embodiment of the present invention is a method of identifyingIAP in cells that includes monitoring a mixture of one or more IAPbinding cargo molecules comprising structure (2), (3), or (5), or dimersthereof, with one or more sample cells for the presence of a detectablelabel from an IAP binding cargo molecule or a change in a detectableproperty of one or more of the IAP binding cargo molecules in themixture. Preferably the detectable property of the IAP binding cargomolecule changes upon formation of a complex between the IAP bindingcargo molecule and the BIR domain of an IAP protein, the IAPs mayinclude but are not limited to XIAP, c-IAP1, c-IAP2, ML-IAP, or survivinin the sample cells. In the sample, the IAP may be bound to a caspase,other proteins like Smac, or combinations of these within the cell.Monitoring may be performed on cells and an IAP binding cargo moleculein a fluid sample, a flowing fluid, or fluids following purification.This invention may be used to detect abnormal expression, over or underexpression, of IAP in cells and the indication of abnormal expressionused to begin a course of treatment of the cells. Preferably the IAPbinding cargo molecule is used to detect overexpression of IAPs incells. The method may further include the act of comparing a change in adetectable property of one or more IAP binding cargo molecules mixedwith one or more control cells to the detectable change in the propertyof the one or more IAP binding cargo molecules mixed with one or moresample cells. The comparison may be related to the amount or activity ofIAP in the sample cells. The method may include the act of combining oneor more IAP binding cargo molecules with one or more cells including butnot limited to sample cells, control cells, or various combination ofthese cells. The monitoring may use the absorption or emission ofradiant energy by the mixture of IAP binding cargo molecules and thecells, including but not limited to magnetic resonance, fluorescence,chemiluminescence, magnetic resonance imaging, and positron emissiontomography. Preferably the change in the detectable property of one ormore of the IAP binding cargo molecules in the mixture chemically, andor physically binding to the IAP in the cells is a change in theintensity of fluorescent emission of the IAP binding molecule. In someembodiments, a change occurs in the fluorescent emission of one or moreIAP binding cargo molecules capable of displacing IAP from caspases orSmac from IAPs in the sample cells.

A method of treating cells of the present invention includes identifyingthe expression of IAP in cells and administering an amount of a cellpermeable IAP-binding cargo molecule or other therapeutic to the cellsto modify the activity of IAP in the cells. The IAP-binding cargomolecule may be formulated with a pharmaceutically acceptable excipient.For example, following identification of abnormal levels of IAP in asample of cells, (optionally by comparison to a control sample ofcells), purified Smac, an IAP binding compound, or an IAP binding cargomolecule, may be added to the cells to induce apoptosis. This inventioncan be used to identify cells in need of treatment, treat the cells, andmonitor the progress of the treatment of the cells having the abnormalIAP levels. The act of identifying cells having abnormal IAP expressionincludes monitoring a mixture of one or more IAP binding cargo moleculeswith one or more sample cells for a change in a detectable property ofone or more of the IAP binding cargo molecules. The detectable propertychanges upon formation of a complex between the IAP binding molecule andIAP in the sample cells.

Another embodiment of the present invention is an article or kit thatincludes packaging material containing a composition of IAP bindingcompound or an IAP binding cargo molecule of formula (2), (3), or (5) ora dimer thereof. The packaging material has a label that indicates howthe IAP binding cargo composition can be used for administration,treatment, or detecting levels of IAP in a sample of cells. The labelmay further indicate how the IAP binding cargo molecule or another IAPbinding cargo molecule included in a pharmaceutical composition can beused to treat cells where an abnormal level of IAP expression isdetermined.

An embodiment of the present invention is a method of selectivelyidentifying neoplastic cells in a mixed population of cells. In themethod a sample of the mixed cell population is contacted with one ormore IAP-binding cargo molecules of formula (2), (3), or (5) or dimersthereof under conditions enabling the IAP-binding cargo molecule to bindIAP within the neoplastic cells and thereby selectively identifying theneoplastic cells. The cells may include but are not limited to culturedcells, cells that are removed from a subject by biopsy, or cells from afluid. The contacting may be performed by introducing the labeledIAP-binding cargo molecule into a tissue sample or a tissue in a livingsubject possessing or suspected of possessing the neoplastic cells. TheIAP-binding cargo molecule can have a dye label cargo portion andpreferably the dye is a fluorogenic dye. The labeled IAP-binding cargomolecule may have an NMR-active nucleus or a contrast agent and theselective identification performed through nuclear magnetic resonance ormagnetic resonance imaging. The labeled LAP-binding cargo molecule mayhave a cargo portion of the molecule that is a radioisotope and wherethe selective identification performed through positron emissiontomography. The IAP binding cargo molecule may be formulated withpharmaceutically acceptable excipients and optionally other therapeuticagents for modifying apoptosis in the sample of cells.

Another embodiment of the present invention is a method of selectivelydamaging or killing neoplastic cells in a mixed population of normal andneoplastic cells. The method includes contacting a sample of the mixedcell population with a cell permeable IAP-binding cargo molecule offormula (2), (3), or (5) or dimers thereof including an agent that isdirectly or indirectly toxic to cells, preferably the cargo portion ofthe molecule is an agent that is directly or indirectly toxic to cells.Under conditions enabling the IAP-binding cargo molecule to bind IAPwithin the neoplastic cells, the agent directly or indirectly exerts itstoxic effect, thereby damaging or killing at least a portion theneoplastic cells. The method may use a toxic agent that is aradioisotope. The method may use a photosensitizing toxic agent and theselective damaging or killing is performed by exposing the cellpopulation to light.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the embodiment's methods, devices, andmaterials are now described. All publications mentioned herein areincorporated by reference. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event occurs and instances where it does not.

The IAP binding molecules of the present invention and pharmaceuticalcompositions containing these compounds can bind to IAP's (Inhibitor ofApoptosis Proteins) such as but not limited to XIAP, c-IAP1, c-IAP2,survin, and LIVIN/ML-IAP. These compounds may include diagnostic ortherapeutic moieties or substituents as part of the binding portion ofthe molecule or linked to the binding portion of the molecule. The IAPbinding cargo molecules may be arbitrarily divided to include an IAPbinding portion and a cargo portion. The IAP binding portion of themolecule interacts with an IAP protein, preferably the BIR domain of anIAP protein, and may be a monomer or dimer. In some embodiments the IAPbinding portion of the molecule interacts with the BIR3 surface grooveof XIAP. The cargo portion of the molecule may be part of the backboneor IAP binding portion of the molecule or the cargo portion may bechemically bonded to the IAP binding portion of the molecule. The cargoportion of the molecule may include but is not limited to structures,moieties, and substituents for imaging, therapeutics, probes, labels, ormarkers. The cargo portion and IAP binding portion of the molecule maybe connected by a chemical bond to the IAP binding portion including butnot limited to amide, ester, or disulfide bonds, chelation, or by alinking group such as diaminobutane or ethylene glycol and its oligomerswhere it is desirable to separate the IAP binding portion of themolecule from the cargo portion of the molecule. One or more atoms inany portion of the IAP binding cargo molecule may be a radioisotope andused for detection or treatment. In other embodiments, the cargo portionmay constitute a second monomer, thereby forming a dimer molecule, via alinking group. The binding portion of the IAP binding compound confersIAP target protein specificity to the molecule and the cargo portion canprovide a functional group to the molecule for monitoring or evaluatingthe location of the molecule or providing a therapeutic to that locationwithin a cell sample or a tissue in a mammal. The IAP binding compoundsmay be used to displace sequestered proteins in cells, for examplecaspase-3, 7 or 9 or Smac interacting with an IAP, so that the releasedprotein can be used to promote apoptosis within the cell. Where thecargo portion of the molecule is linked to the IAP binding portion, thecargo portion may be bonded or chemically linked to any portion of theIAP binding portion of the molecule so that it does not adversely affectIAP binding, cell permeance or transfection into cells. While chemicalinteraction between the IAP binding portion and the cargo portion of themolecule may occur, the molecule is made so that the molecule's cellpermeance, its IAP binding property, and function of the cargo portionare not adversely affected by their combination. The suitability of anyIAP binding cargo molecule made by the method disclosed may be testedagainst fluorescently labeled peptide [AbuRPF—K(5-Fam)-NH₂] in cellssuch but not limited to renal cell carcinoma, non-small cell lungcancer, HeLa cells, or others known to overexpress IAP, or other cellshaving a proliferation disorder. The IAP binding molecules of thepresent invention are capable of permeating cells of interest, bindingto IAP in the cells, and optionally delivering the cargo to the cells.

Embodiments of compounds of structure (2), (3), (5) include2-substituted pyrrolidine-1-carbonyls, or 2, 4-independently substitutedpyrrolidine-1-carbonyls that have a K_(d) as determined by methodsdescribed, for example, in Example 1 of less than 100 micromolar,preferably less than 1 micromolar, and even more preferably less than0.1 micromolar.

The pharmaceutically active compounds of the invention are sometimesreferred to herein as drugs, to highlight their therapeutic utility inpromoting apoptosis by binding IAPs. However, another embodiment of theinvention utilizes the compounds as diagnostic agents, for detection ofIAP in vitro, in situ or in vivo, or for IAP binding assays. In theseembodiments, the compounds of the invention are detectably labeled, forexample, with a fluorophore. Another embodiment of the inventionutilizes the compounds as targeting agents, i.e., by incorporating intotheir structure tumor cell-killing or other anti-tumor or therapeuticagents, such as radionuclides. Accordingly, drugs refer topharmaceutically/biologically active (i. e., IAP-binding) compounds ofthe invention, for use as therapeutic, prophylactic, or diagnosticagents.

The term heteroatom refers to nitrogen, oxygen, sulfur, other atoms orgroups where the nitrogen, sulfur and other atoms may optionally beoxidized, and the nitrogen may optionally be quaternized. Any heteroatomwith unsatisfied valences is assumed to have hydrogen atoms sufficientto satisfy the valences. In some embodiments, for example where theheteroatom is nitrogen and includes a hydrogen or other group to satisfythe valance of the nitrogen atom, replacement of the nitrogen in asimilar structure by another heteroatom, for example by oxygen, willresult in the hydrogen or group previously bonded to the nitrogen to beabsent. The term heteroatom may include but is not limited to forexample —O—, —S—, —S(O)—, —S(O)₂—, —N—, —N(H)—, and —N(C₁-C₆ alkyl).

The term alkyl refers to a saturated straight, branched, or cyclichydrocarbon having from about 1 to about 30 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein). Lower alkyl group refers to a saturated straight,branched, or cyclic hydrocarbon having group of 1 to 10 carbon atoms,more preferably 1 to 5 carbon atoms and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein.Alkyl groups include, but are not limited to methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopropyl,methylcyclopropyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl,cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl,and 2,3-dimethylbutyl.

The term substituted alkyl refers to a saturated straight, branched, orcyclic hydrocarbon having from about 1 to about 30 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein) having from 1 to 5 substituents. Substituted loweralkyl group refers to a saturated straight, branched, or cyclichydrocarbon of 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein) having from 1 to 5 substituents. Substitutedalkyl radicals and substituted lower alkyl groups can have from 1 to 5substituents including but not limited to alkoxy, substituted alkoxy,acylamino, thiocarbonylamino, acyloxy, amino, amidino, alkylamidino,amidalkyl (such as —CH₂C(═O)NH₂ or —CH₂CH₂C(═O)NH₂), thioamidino,acylalkylamino, cyano, halogen atoms (F, Cl, Br, I) to give halogenatedor partially halogenated alkyl groups, including but not limited to—CF₃, —CF₂CF₃, —CH₂CF₂Cl and the like, hydroxy, nitro, carboxyl,carboxylalkyl, carboxylheterocyclic, carboxyl-substituted heterocyclic,cycloalkyl, guanidino, heteroaryl, aryl, heterocyclic, alkylamino,dialkylamino, or optionally substituted versions of any of theaforementioned groups.

The term alkylene radical as used herein includes reference to adi-functional saturated branched or unbranched hydrocarbon radicalcontaining from 1 to 30 carbon atoms, and includes, for example,methylene (—CH₂—), ethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—),2-methylpropylene (—CH₂CH(CH₃)CH₂—), hexylene (—(CH₂)₆—), and the like.Lower alkylene includes an alkylene group of 1 to 10, more preferably 1to 5, carbon atoms.

Substituted alkylene radicals includes reference to a di-functionalsaturated branched or unbranched alkylene radical or group having 1-30carbon atoms and having from 1 to 5 substituents. Lower substitutedalkylene radicals refer to a substituted alkylene radical group, having1-10 carbon atoms, preferably having 1-5 carbon atoms, and having from 1to 5 substituents. Substituents can include but are not limited to thosefor the alkyl groups.

The term alkenyl radical as used herein includes reference to abranched, cyclic hydrocarbon, or unbranched hydrocarbon radical of 2 to30 carbon atoms containing at least one carbon-carbon double bond, suchas ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, t-butenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl andthe like. The term lower alkenyl includes an alkenyl group of 2 to 10carbon atoms, preferably 2 to 5 carbon atoms, containing at least onecarbon-carbon double bond. The one or more carbon-carbon double bondsmay independently have a cis or trans configuration. Substituted alkenylradical refers to an alkenyl radical or lower alkenyl group having from1 to 5 substituents that can include but are not limited to those forthe alkyl groups.

The term alkenylene radical includes reference to a difunctionalbranched or unbranched hydrocarbon radical or group containing from 2 to30 carbon atoms and at least one carbon-carbon double bond. “Loweralkenylene” includes an alkenylene group of 2 to 10, more preferably 2to 5, carbon atoms, containing one carbon-carbon double bond.Substituted alkenylene radical refers to an alkenylene radical or loweralkenyl group having from 1 to 5 substituents that can include but arenot limited to those for the alkyl groups.

The term alkynyl radical or group refers to straight or branched chainhydrocarbon radical having 2 to 12 carbon atoms and at least one triplebond, some embodiments include alkynyl groups of 2 to 6 carbon atomsthat have one triple bond. A substituted alkynyl will contain one, two,or three substituents as defined for substituted alkyl groups.Alkynylene includes reference to a difunctional branched or unbranchedhydrocarbon chain containing from 2 to 12 carbon atoms and at least onecarbon-carbon triple bond; some embodiments include an alkynylene groupsof 2 to 6 carbon atoms with one triple bond. A substituted alkynylenewill contain one, two, or three substituents as defined for substitutedalkyl groups.

As used herein, “halo” or halogen refers to any halogen, such as I, Br,Cl or F. As used herein, “cyano” refers to the —C≡N group.

The term aryl radical or group refers to an optionally substituted, monoor bicyclic aromatic ring radicals having from about 5 to about 14carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 6 to about 10carbons being preferred. Non-limiting examples or aryl groups include,for example, phenyl and naphthyl. A substituted aryl group will containone or more substituents as defined for substituted alkyl groups.

Aralkyl radical refers to alkyl radicals bearing an aryl substituent andhave from about 6 to about 20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein),with from about 6 to about 12 carbon atoms being preferred. Aralkylgroups can be optionally substituted. Non-limiting examples include, forexample, benzyl, naphthylmethyl, diphenylmethyl, triphenylmethyl,phenylethyl, and diphenylethyl. A substituted arylalkyl group willcontain one or more substituents on the aryl or alkyl group as definedfor substituted alkyl groups.

Cycloalkylaryl radical or group refers to a cycloalkyl radical fused toan aryl group, including all combinations of independently substitutedalkyl cycloalkylaryls, the cycloalkyl and aryl group having two atoms incommon. Examples of fused cycloalkylaryl groups used in compounds of thepresent invention may include 1-indanyl, 2-indanyl,1-(1,2,3,4-tetrahydronaphthyl), and the like. Tetrahydronaphthyl morespecifically refers to those univalent radicals or groups derived fromfused polycyclic hydrocarbons including all combinations ofindependently substituted alkyl tetrahydronaphthyls. These radicals mayhave a point of attachment at (C₁) or equivalently (C₄) in structure(11), or position labeled (C₂) and equivalently (C₃) in structure (11a).The chiral carbon atoms C₁₋₄ in tetrahydronaphthlene and its alkylsubstituted derivatives may have either an (R) or (S) configuration.

Cycloalkyl radical or group more specifically includes reference to amonovalent saturated carbocyclic alkyl radical consisting of one or morerings in their structures and having from about 3 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 3 to about 7 carbonatoms being preferred. Multi-ring structures may be bridged or fusedring structures. The rings can optionally be substituted with one ormore of the substituents for the alkyl groups. Examples of cycloalkylgroups include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclooctyl, and adamantyl. A substitutedcycloalkyl group will contain one or more substituents as defined forsubstituted alkyl groups.

Cycloalkylalkyl radical more specifically refers to alkyl radicalsbearing an cycloalkyl substituent and having from about 4 to about 20carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 6 to about 12carbon atoms being preferred and can include but are not limited tomethylcyclopropyl, methylcyclohexyl, isopropylcyclohexyl, andbutyl-cyclohexyl groups. Cycloalkylalkyl radical or group can beoptionally substituted with one or more substituents for the alkylgroups including but not limited to hydroxy, cyano, alkyl, alkoxy,thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, alkylamino anddialkylamino.

The term acyl refers to an alkyl, substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl group asdefined above bonded through one or more carbonyl —C(═O)— groups to givea group of formula —C(═O)R where R is the substituted alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclylgroup. When the term acyl is used in conjunction with another group, asin acylamino, this refers to the carbonyl group {—C(═O)} linked to thesecond named group. Thus, acylamino or carbamoyl refers to —C(═O)NH₂,acylalkylamino can refer to groups such as —C(═O)NR′R″ where R′ and R″can be H or alkyl. Amidalkyl refers to groups such as —CH₂C(═O)NH₂,—CH₂CH₂C(═O)NH₂) and more generally —(CH₂)_(p)C(═O)NH₂. Carboxy refersto the radical or group —C(═O)OH, carboxyalkyl refers to the groups suchas —(CH₂)_(p)C(═O)OH, alkyl carboxyalkyl refers to groups such as(—C(═O)O-(alkyl)), and alkoxycarbonyl or acylalkoxy refers to a(—C(═O)O-(alkyl)) group, where alkyl is previously defined. As usedherein aryoyl or acylaryl refers to a (—C(═O)(aryl)) group, wherein arylis as previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl. Acetyl refers to the group CH₃C(═O)— and may be abbreviatedby the term “Ac” as is Table 5. Formyl refers to the radical or groupHC(═O)—. In the aforementioned group p can be independently the integer0, 1, 2, or 3.

Aryloxy radical refers to optionally substituted mono or bicyclicaromatic radical having from about 5 to about 14 carbon atoms and an(aryl-O—) radical group wherein aryl is as previously defined. Sucharyloxy radicals include but are not limited to that illustrated by theradical of formula (12). Optional substituents on the aryl ring in thearyloxy radical may include but are not limited to hydrogen, alkyl,halogen, hydroxy, alkoxy, alkoxycarbonyl or other substituents.Embodiments of IAP binding compounds of the present invention caninclude an optionally aryloxy group like the phenoxy radical linked tothe pyrrolidine ring as illustrated but not limited to compounds inTable 5. Some embodiment of IAP binding compounds include include aphenoxy radical where the K_(d) as determined by methods described, forexample, in Example 1 is less than about 0.1 micromolar.

The terms “alkoxy” and “alkoxyl” refer to an optionally substituted(alkyl-O—) radical or group wherein alkyl is as previously defined.Exemplary alkoxy radicals or groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, cyclopropyl-methoxy, and heptoxy. Alkoxy radicalscan also include optionally substituted alkyl in the alkylO— group.Alkoxy can include including optionally substituted aryl groups aspreviously defined and illustrated by the non-limiting radical offormula (13). A “lower alkoxy” group refers to an optionally substitutedalkoxy group containing from one to five carbon atoms. “Polyether”refers to a compound or moiety possessing multiple ether linkages, suchas, but not limited to, polyethylene glycols or polypropylene glycols.“Polyalkylethers” refers to alkyls interconnected by or otherwisepossessing multiple ether linkages as illustrated by the non-limitingstructure of formula (85) in Scheme VIII of Example 16. “Arylalkyloxy”means an arylalkyl-O— group in which the arylalkyl group is aspreviously described. Exemplary arylalkyloxy groups include benzyloxy(C₆H₅CH₂O—) radical (BnO—), or 1- or 2-naphthalenemethoxy. Optionalsubstituents on the aryl ring in the benzyloxy radical may include butare not limited to hydrogen, alkyl, halogen, hydroxy, alkoxy, andalkoxycarbonyl or other substituents as defined for the alkyl group.

Arylamino radical refers to optionally substituted mono or bicyclicaromatic radical having from about 5 to about 14 carbon atoms and an(—NH(aryl)) radical group wherein aryl can be optionally substituted aspreviously defined for alkyl. Optional substituents on the aryl ring inthe arylamino radical may include but are not limited to hydrogen,alkyl, halogen, hydroxy, alkoxy, and alkoxycarbonyl. A example of anarylamino group is the anilino radical or group. Amino refers to an —NH₂group and alkylamino refer to a radical (—NH R′) group wherein R′ is H,alkyl, cycloalkyl, aryl, heteroaryl, aralkyl or optionally substitutedversions of these as previously defined. Exemplary alkylamino radicalgroups include methylamino, ethylamino, n-propylamino, i-propylamino,n-butylamino, and heptylamino. The benzylamino radical refers to thearylamino group C₆H₅CH₂NH—, the aryl group may have optionalsubstituents including but are not limited to hydrogen, alkyl, halogen,hydroxy, alkoxy, and alkoxycarbonyl or other substituents.

Dialkylamino includes reference to a radical (—NR′R″), wherein R′ and R″can be each independently be an H, alkyl, cycloalkyl, aryl, heteroaryl,aralkyl or optionally substituted versions of these as previouslydefined. Examples of dialkylamino radicals include, but are not limitedto, dimethylamino, methylethylamino, diethylamino,di(1-methylethyl)amino, and the like.

Heteroaryl includes reference to a monovalent aromatic radical or grouphaving one or more rings incorporating one, two or three heteroatomswithin the ring (chosen from nitrogen, oxygen, or sulfur). Theseheteroaryls can optionally have hydrogen atoms substituted with one ormore other substituents. Examples of these heteroaryl radicals includeoptionally substituted benzofurans, benzo[b]thiophene 1-oxide, indoles,2- or 3-thienyls or thiophenyls, thiazoyls, pyrazines, pyridines, or thestructures of (4a) or (4b). For example, the structure of formula (E10)with derivatives listed in Table 9, can include a fused ring R₁₀ with aheteroatom X₂ (N, O, or other) and substituents such as R₁₁ or R′₁₁including but not limited to hydrogen, halogens, optionally substitutedheteroaryls like pyridine, benzofuran, indoles, thiazoyl, pyrazine, analkoxyheteroaryl like methoxy pyridine, or other groups.

The terms heteroalkyl, heteroalkylene, heteroalkenyl, heteroalkenylene,heteroalkynyl, and heteroalkynlene include reference to alkyl, alkylene,alkenyl, alkenylene, alkynyl, and alkynlene radicals or groups, in whichone or more of the carbon atoms have been replaced or substituted withatoms such as but not limited to single or multiply bonded nitrogen,sulfur, oxygen, or these atoms having one or more hydrogens to satisfythe valancy requirements of the atom. Such substitutions can be used toform molecules having functional groups including but not limited toamines, ethers, and sulfides. A non-limiting example of a heteroalkynylgroup is illustrated by the group —CH(Me)OCH₂C≡CH.

Heterocycloalkyl radical include reference to a monovalent saturatedcarbocyclic radical or group consisting of one or more rings,incorporating one, two or three heteroatoms (chosen from nitrogen,oxygen or sulfur), which can optionally be substituted with one or moresubstituents.

Heterocycloalkenyl includes reference to a monovalent unsaturatedcarbocyclic radical consisting of one or more rings containing one ormore carbon-carbon double bonds where carbon atoms are replaced orsubstituted for by one, two or three heteroatoms within the one or morerings, the heteroatoms chosen from nitrogen, oxygen, or sulfur, theheterocycloalkenyl can optionally be substituted with one or moresubstituents.

Various groups used in the molecules of the present invention can haveone or more hydrogens atoms substituted for chemical moieties or othersubstituents. Substituents may include but are not limited to halo orhalogen (e. g, F, Cl, Br, I), haloalkyls such as —CF₃, —CF₂CF₃, —CH₂CF₃and the like, thioalkyl, nitro, optionally substituted alkyl,cycloalkyl, aralkyl, aryl, heteroaryls like benzofurans, indoles,thienyls, thiophenyls, thiazoyls, pyrazines, pyridines, alkoxy pyridine,hydroxy (—OH), alkoxy (—OR), aryloxy, alkoxyheteroaryl, cyano (—CN),carbonyl —C(═O)—, carboxy (—COOH) and carboxylate salts;—(CH₂)_(p)C(═O)OH, groups or radicals —(CH₂)_(p)C(═O)O(alkyl), and—(CH₂)_(p)C(═O)NH₂ where p is independently the integer 0, 1, 2, or 3;sulfonates such as but not limited to tosyl, brosyl, or mesyl; sulfone,imine, or oxime groups, groups like —(C═O)Oalkyl, aminocarbonyl orcarbamoyl —(C═O)NH₂), —N-substituted aminocarbonyl —(C═O)NHR″, amino,alkylamino (—NHR′) and dialkylamino (—NHR′R″). In relation to theaforementioned amino and related groups, each moiety R′ or R″ can be,independently include of H, alkyl, cycloalkyl, aryl, heteroaryl, aralkylor optionally-substituted alkyl, cycloalkyl, aryl, heteroaryl, aralkyl.Where one or more hydrogens atoms are substituted for chemical moietiesor other substituents, the substituents can be chosen so that IAPbinding compounds of formula (2), (3), or (5) that contain them have aK_(d) as measured by methods described, for example, in Example 1 ofless than about 100 micromolar, in some embodiments have a K_(d) of lessthan 1 micromolar, and in other embodiments have a K_(d) of less than0.1 micromolar. In embodiments of where one or more hydrogens atoms aresubstituted for chemical moieties or other substituents, thesubstituents can be chosen so that IAP binding compounds of formula (2),(3), or (5) that contain them have an EC₅₀ as measured by methodsdescribed, for example, in Example 2 of less than about 0.5 micromolar,in some embodiments have an EC₅₀ of less than about 0.06 micromolar. Inembodiments of where one or more hydrogens atoms are substituted forchemical moieties or other substituents, the substituents can be chosenso that IAP binding compounds of formula (2), (3), or (5) that containthem have a binding constant K_(d) of less than about 1 micromolar,preferably less than 0.1 micromolar and an EC₅₀ of less than about 1micromolar, preferably less than about 0.5 micromolar, and in someembodiments an EC₅₀ of less than about 0.06 micromolar.

Amino acids can be used in the IAP binding compound compounds of thisinvention and may include the 20 naturally-occurring amino acids, knownartificial amino acids such as beta or gamma amino acids, and aminoacids containing non-natural side chains, and/or other similar monomerssuch as hydroxyacids. Preferably the amino acids used in the IAP bindingcompounds of the present invention are the 20 naturally-occurring aminoacids. The amino acids or artificial amino acids are chosen with theeffect that the corresponding IAP binding compound binds IAPs, andpreferably binds the BR domain of an IAP, and the resulting IAP bindingcompound is permeable to the cell. A non-limiting example of such anamino acid includes the use of Abu (2-aminobutyric acid) as an aminoacid in the IAP binding cargo molecule. Where the molecule of structure(2), (3), or (5) includes amino acids, it is preferred that theN-terminal amino acid is Ala or Abu.

Where one or more chiral centers exist in an amino acid, artificialamino acid, or atom of an IAP binding compound of structure (2), (3), or(5), any of the enantiomers, D or L and more generally R or Sconfiguration, or diastereoisomers may optionally be used in the IAPbinding compound.

When any variable occurs more than one time in any constituent or in anyformula, its definition in each occurrence is independent of itsdefinition at every other occurrence. Combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

It is believed the chemical formulas and names used herein correctly andaccurately reflect the underlying chemical compounds. However, thenature and value of the present invention does not depend upon thetheoretical correctness of these formulae, in whole or in part. Thus itis understood that the formulas used herein, as well as the chemicalnames attributed to the correspondingly indicated compounds, are notintended to limit the invention in any way, including restricting it toany specific tautomeric form or to any specific optical; or geometricisomer, except where such stereochemistry is clearly defined.

In embodiments of compounds of structure (2), (3), or (5) substituentssuch as A₁, A₂, R_(1a), and R_(1b), R₂, X₁, Y, Z, M, G, or R₁₀, may bechosen independently so that compounds of structure (2), (3), or (5) arenot a tripeptide or a tetrapeptide of amino acids, for example AVPI orAVP.

Some embodiments of the compounds of structure (2), when A₂ is H, X₁ is—NH—, J is —CH—, and n is 0 for R₂, can be depicted by structure (3):

In some embodiments of compounds of structure (3), A₁ is H, lower alkyl,or optionally-substituted lower alkyl group, an N-acyl derivative likeacylamino or acylalkylamino, t-butoxycarbonyl, acetyl, formyl,carbamoyl, alkylene, or other; R_(1a) and R_(1b) are separately H, loweralkyl, optionally-substituted lower alkyl, lower alkylene, optionallysubstituted lower alkylene group; or A₁ together with either R_(1a) orR_(1b) form an optionally substituted heterocycloalkyl group of 3 to 6atoms as illustrated by some of the non-limiting embodiments ofcompounds in Table 5.

In IAP binding compounds of structure (3), Y can be H, an alkyl group,an alkyl group of 1 to 10 carbon atoms, a branched alkyl group of 1 to10 carbon atoms, an alkynyl group, heteroalkynyl, a cycloalkyl group of3 to 7 carbon atoms, aryl, heteroaryl, arylalkyl; optionally-substitutedversions of the aforementioned groups such as optionally substitutedalkyl group, optionally substituted alkyl group of 1 to 10 carbon atoms,an optionally substituted branched alkyl group of 1 to 10 carbon atoms,an optionally substituted alkenyl group, an optionally substitutedalkynyl group, an optionally substituted heteroalkynyl group, anoptionally substituted cycloalkyl group or 3 to 7 carbon atoms, anoptionally substituted aryl group, an optionally substituted heteroarylgroup, an optionally substituted arylalkyl group; in some embodimentsthe substituent or moiety for the aforementioned substituted groupincludes one or more hydroxy groups; or Y together with Z, M, G, or R₁₀can for an optionally substituted carbocyclic ring, or an optionallysubstituted heterocyclic ring containing 1 to 5 heteroatoms, where Y islinked to Z, M, G, or R₁₀; preferably Y is linked to Y, M, G, or R₁₀ byany number of atoms up to about 20 atoms. A non-limiting example of sucha heterocyclic ring is illustrated by the IAP binding molecule havingthe structure of formula (E12-2) in Example 12.

In IAP binding compounds of structure (3), Z can be H, alkyl, hydroxy,amino, alkylamino, diakylamino, alkoxy, cycloalkyl, cycloalkyloxy, aryl,heteroaryl, aryloxy, heteroaryloxy; optionally substituted versions ofthese groups; optionally substituted versions of these groups includingone or more hydroxyl groups; or Z together with Y, M, G, or R₁₀ can forman optionally substituted carbocyclic ring, or an optionally substitutedheterocyclic ring containing 1 to 5 heteroatoms, where Z is linked to Y,M, G, or R₁₀; preferably Z is linked to Y, M, G, or R₁₀ by any number ofatoms up to about 20 atoms. In some embodiments, Z is an alkyl, hydroxy,amino, alkylamino, dialkylamino, alkoxy, cycloalkyl, cycloalkyloxy,aryl, heteroaryl, aryloxy, or heteroaryloxy group. The stereochemistryof the Z substituent or group may be indicated using a bold wedged bondto show a Z group coming out of the plane of the page or a dashed wedgedbond to show a Z group behind the plane of the page. In embodiments of(3), the IAP binding molecule may have a structure where the Z group isdirected out of the plane of the page, the IAP binding molecule may havea structure where the Z group is directed behind the plane of the page,or the IAP binding molecule may include a mixture where the Z group is acombination of these.

In IAP binding compounds of structure (3), M can be anoptionally-substituted alkyl, alkenyl, or alkynyl group; M can be anoptionally-substituted alkyl, alkenyl, or alkynyl group of 1 to 5 carbonatoms; M can be an optionally-substituted alkylene, alkenylene, oralkynylene group; or an optionally-substituted alkylene, alkenylene, oralkynylene group of 1 to 5 carbon atoms. In some embodiments, forexample but not limited to IAP binding compounds of structure (E6) or(E14), M is a diradical alkylene like the methylene group linked at oneend of the methylene group the 2 position of the pyrrolidine ring and atthe other end of the methylene group to an aryl group, a heteroarylgroup, or an optionally substituted heteroaryl group like a benzofuranor indole group, or optionally substituted versions of groups ofstructure (4a-d).

In IAP binding compounds of structure (3), G can be absent (a bond) asin the non-limiting examples of compounds of structure (E6) or (E14), orG can be a heteroatom including but not limited to —O—; —NH—; —(C═O)—;—S(O)_(t)— where t can be the integer 0, 1, or 2; —NR₁₈—; —NCOR₁₈—; or—NS(O)_(x)R₁₈— where x can be the integer 0, 1, or 2, and R₁₈ can belower alkyl, optionally-substituted lower alkyl, or cycloalkyl or R₁₈can be contained within an optionally substituted carbocyclic, oroptionally substituted heterocyclic ring containing 1 to 5 heteroatoms,where R₁₈ is linked to Z, M, or R₁₀, preferably R₁₈ is linked to Z, M,or R₁₀, by any number of atoms up to about 20 atoms.

In IAP binding compounds of structure (3), R₁₀ can be an aryl, aheteroaryl group, a fused aryl, or a fused heteroaryl group. In someembodiments, for example but not limited to compounds in Table 5, R₁₀can be a substituted aryl, a substituted heteroaryl group, a substitutedfused aryl, or a substituted fused heteroaryl group. In some embodimentsR₁₀ can be any one of structures (4a), (4b), (4c), or (4d):

where X₂ is a heteroatom in structures (4a) or (4b) or X₂ is acarbon-carbon bond in structures (4c) or (4d), and independently groupsR₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ can be H, halogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl, hydroxyl, alkoxy,polyalkylether, amino, alkylamino, dialkylamino, alkyloxyalkyl,sulfonate, aryloxy, heteroaryloxy, other substituents or optionallysubstituted versions of these groups. In embodiments where R₁₁ or R′₁₁is a heteroaryl group, it can be a 2- or 3-thienyl SC₄H₃— (thiophenyl)group, pyridine, pyrazine or optionally substituted versions of these.In some embodiments R₁₂ can be an aryl or a heteroaryl group, such asbut not limited to benzofuran, indole, benzo[b]thiophene-1-oxide orbenzo[b]thiophene-1,1-dioxide or optionally substituted versions ofthese. Where X₂ is either —O— or —S(O)_(k)—, for the integer k which canindependently be 0, 1, or 2, R₁₂ is absent. Independently R₁₁, R′₁₁,R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ can be H, optionally-substitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, hydroxyl, alkoxy,polyalkylether, carboxyalkyl, alkyl carboxyalkyl, amino, alkylamino,dialkylamino, alkyloxyalkyl, aryloxy, or heteroaryloxy. IndependentlyR₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ can be acyl or acetylgroups, carboxylate, sulfonate, sulfone, imine, or oxime groups; orgroups R₁₁, R′₁₁, R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ can be containedwithin a carbocyclic ring, or a heterocyclic ring containing 1 to 5heteroatoms, and linked to groups at position Y, Z, M, G, R_(u), R′₁₁,R₁₂, any of R₁₃₋₁₇, or any of R₁₄₋₁₇ preferably these groups are linkedby any number of atoms up to about 20 atoms. In some embodiments R₁₀ canbe cycloalkyl, aryl, heterocycloalkyl, heterocycloalkenyl, heteroaryl,or optionally substituted embodiments of these. In some embodiments R₁₀can be the group:

where R₃, R′₃, R₄, R₅, R′₅, can each independently be substituents likeH, alkyl, cycloalkyl, alkylene, aryl, heteroaryl, alkoxy optionallysubstituted alkyl, cycloalkyl, alkylene, aryl, heteroaryl, halo, cyano,—(CH₂)_(p)C(═O)OH, —(CH₂)_(p)C(═O)O-alkyl, —(CH₂)_(p)C(═O)NH₂, p isindependently the integer 0, 1, 2, or 3. For example, in the IAP bindingcompound of structure (E4) where M is alkenylene, G is a heteroatom like—O—, R₁₀ can be an optionally substituted aryl group having substituentsfor example but not limited to hydrogen, chloro, bromo, alkyl, alkylene,alkoxy, or combinations of these.

Some embodiments of IAP binding compounds of formula (3) includecompounds of structure (5) where:

A₁ is H, or lower alkyl, or A₁ and R_(1b) together form a ring of 3-5atoms. In these embodiments, R_(1a) is H and R_(1b) can be lower analkyl group, or together with A₁ forms a ring of 3 to 5 atoms.

In embodiments of IAP binding compounds of structure (5), Y can be analkyl group, an alkyl group of 1 to 10 carbon atoms, a branched alkylgroup of 1 to 10 carbon atoms, an alkynyl group, heteroalkynyl, acycloalkyl group of 3 to 7 carbon atoms, optionally substituted versionsof the aforementioned groups, and/or hydroxy substituted versions of theaforementioned groups, or Y together with Z_(1a), Z_(1b), or R₁₀ forms acarbocyclic ring, or a heterocyclic ring containing 1 to 5 heteroatoms,where Y is linked to Z_(1a), Z_(1b), or R₁₀; preferably Y is linked toZ_(1a), Z_(1b), or R₁₀ by any number of atoms up to about 20 atoms;

In embodiments of IAP binding compounds of structure (5), Z_(1a) andZ_(1b) can independently be an H, hydroxy, amino, alkylamino,diakylamino, alkoxy, aryloxy, or heteroaryloxy group; or Z_(1a), orZ_(1b), together with Y or R₁₀ form a carbocyclic ring, or aheterocyclic ring containing 1 to 5 heteroatoms, where Z_(1a) or Z_(1b),is linked to Y or R₁₀; preferably Z_(1a) or Z_(1b), is linked to Y orR₁₀ by any number of atoms up to about 20 atoms;

In embodiments of IAP binding compounds of structure (5), M can be anoptionally-substituted alkyl or an optionally-substituted alkylene groupof 1 to 5 carbon atoms. In these structures, G can be absent (a bond),or a heteroatom including —O—; —NH—; —(C═O)—; —NR₁₈—; —NCOR₁₈—; or—NS(O)_(x)R₁₈— where x=0, 1, or 2, and R_(1b) is lower alkyl,optionally-substituted lower alkyl group.

The IAP binding compounds or molecules of structure (2), (3), or (5) canbe prepared by various processes, which are described by thenon-limiting Schemes in the Examples and which also form part of thesubject matter of the present invention. These IAP binding molecules maybe prepared from molecules of structure (6a) or (6b):

where Y, M, Z, G, R₂ and R₁₀ are previously described and where R′ andR″ can be H, or a protecting group. The stereochemistry of the Z or Msubstituent or group may be indicated using a bold wedged bond to showeither group coming out of the plane of the page or a dashed wedged bondto show a Z or M group behind the plane of the page. In embodiments ofstructure (6a) or (6b), the IAP binding molecule may have a structurewhere the Z or M group is directed out of the plane of the page, the IAPbinding molecule may have a structure where the Z or M group is directedbehind the plane of the page, or the IAP binding molecule may include amixture where the Z or M group includes a combination of these.Structures of formula structure (6a) or (6b) may be prepared fromcompounds such as but not limited to pyrrolidines like (1) in Scheme Ior (29) in Scheme IVa using the procedures or similar processesdescribed herein. The structures of formula structure (6a) or (6b) maybe further reacted to yield IAP binding molecules of structure (2), (3),or (5) by treatment with an N-Boc-amino acid or other suitable aminecontaining moiety that includes A₁, A₂, R_(1a), or R_(1b) orcombinations of these as described herein.

Certain acidic or basic compounds of the present invention may exist aszwitterions. All forms of the compounds, including free acid, free baseand zwitterions, are contemplated to be within the scope of the presentinvention. It is well known in the art that compounds containing bothamino and carboxyl groups often exist in equilibrium with theirzwitterionic forms. Thus, any of the compounds described hereinthroughout that contain, for example, both amino and carboxyl groupsalso include reference to their corresponding zwitterions.

In any of the above teachings, an IAP binding cargo molecule or otherIAP binding compound of the invention may be either a compound of one ofthe formulae herein described, or a stereoisomer, prodrug,pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate,or isomorphic crystalline form thereof.

Pharmaceutically acceptable salt refers to those salts of the IAPbinding cargo molecules and IAP binding compounds of structure (2), (3),or (5) or dimers thereof which retain the biological effectiveness andproperties of the free bases or free acids, cell permeation and IAPbinding, and which are not biologically or otherwise undesirable. If thecompound exists as a free base, the desired salt may be prepared bymethods known to those of ordinary skill in the art, such as treatmentof the compound with an inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike; or with an organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. If the compoundexists as a free acid, the desired salt may also be prepared by methodsknown to those of ordinary skill in the art, such as the treatment ofthe compound with an inorganic base or an organic base. Salts derivedfrom inorganic bases include, but are not limited to, the sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike.

The IAP binding cargo molecules or their pharmaceutically acceptablesalts may include pharmaceutically acceptable solvent molecules withintheir crystal lattice. Where the solvent is water, the compounds mayform hydrates, in the case of other solvents and in particular organicsolvents such as but not limited to ethanol the compounds may formsolvates. The IAP binding cargo molecules or IAP binding compounds ofstructure (2), (3), or (5) and its homologs may be formulated, isolated,or purified as solvates.

The compounds employed in the methods of the present invention may existin prodrug form. Prodrug includes any covalently bonded carriers whichrelease the active parent drug, for example, as according to formula (2)or other formulas or compounds employed in the methods of the presentinvention in vivo when such prodrug is administered to a mammaliansubject. Since prodrugs are known to enhance numerous desirablequalities of pharmaceuticals (e.g. solubility, bioavailability,manufacturing, etc.) the compounds employed in the present methods may,if desired, be delivered in prodrug form. Thus, the present inventioncontemplates methods of delivering prodrugs. Prodrugs of the compoundsemployed in the present invention, for example formula (2) may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compound.

Accordingly, prodrugs include, for example, compounds described hereinin which a hydroxy, amino, or carboxy group is bonded to any group that,when the prodrug is administered to a mammalian subject, cleaves to forma free hydroxyl, free amino, or carboxylic acid, respectively. Examplesinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol and amine functional groups; and alkyl,carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, n-propyl,iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl,benzyl, and phenethyl esters, and the like.

The compounds employed in the methods of the present invention may beprepared in a number of ways well known to those skilled in the art. Thecompounds can be synthesized, for example, by the methods described inthe specification and example, or variations thereon as appreciated bythe skilled artisan. All processes disclosed in association with thepresent invention are contemplated to be practiced on any scale,including microgram, milligram, gram, multi-gram, kilogram,multi-kilogram or commercial industrial scale.

IAP binding compounds of structure (2), (3), or (5) can be mixed orcombined with pharmaceutically acceptable excipients or treated bylyophilization. These pharmaceutical compositions may be administeredtopically, locally or systemically to a sample of cells, a tissue, orpatient. Topical or local administration can allow for greater controlof application of the pharmaceutical composition. The IAP bindingmolecules or compounds including structure (2), (3), or (5) singularlyor in combination, can be mixed with an appropriate pharmaceuticalcarrier prior to administration. Examples of generally usedpharmaceutical carriers and additives that can be used to formpharmaceutical, diagnostic, or therapeutic composition of the IAPbinding molecules may include but are not limited to conventionaldiluents, binders, lubricants, coloring agents, disintegrating agents,buffer agents, isotonizing agents, preservants, anesthetics and thelike. Pharmaceutical carriers that may be used include but are notlimited to water, saline, ethanol, dextran, sucrose, lactose, maltose,xylose, trehalose, mannitol, xylitol, sorbitol, inositol, serum albumin,gelatin, creatinine, polyethylene glycol, non-ionic surfactants (e.g.polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hardenedcastor oil, sucrose fatty acid esters, polyoxyethylene polyoxypropyleneglycol) and similar compounds. Pharmaceutical carriers and excipients aswell as IAP binding molecules may also be used in combination.

Stereoisomers are compounds having identical molecular formulae andnature or sequence of bonding but differing in the arrangement of theiratoms in space and include optical and geometrical isomers. Compounds ofthe present invention, or their pharmaceutically acceptable salts, canhave one or more asymmetric carbon atoms or other asymmetric atoms intheir structure, and may therefore exist as single stereoisomers,enantiomers, diastereoisomers, racemates, and mixtures of enantiomers ordiastereomers. These compounds may also include geometric isomers. Allsuch stereoisomers, racemates and mixtures thereof the IAP bindingcompounds and IAP binding cargo molecules of the present invention areintended to be within the scope of this invention unless the specificstereochemistry or isomeric form is specifically indicated. It is wellknown in the art how to prepare and isolate such optically active forms.For example, mixtures of stereoisomers may be separated by standardtechniques including, but not limited to, resolution of racemic forms,normal, reverse-phase, and chiral chromatography, preferential saltformation, recrystallization, and the like, or by chiral synthesiseither from chiral starting materials or by deliberate synthesis oftarget chiral centers.

IAP binding molecules of structures (2), (3), or (5) that contain chiralcenters and the molecules can be in the form of a single enantiomer oras a racemic mixture of enantiomers. In some cases, i.e., with regard tothe structure of certain specific IAP binding molecules, chirality(i.e., relative stereochemistry) of substituents or groups is indicatedin the structure using a bold wedged bond to indicate a substituentcoming out of the plane of the page and a dashed wedged bond to indicatea substituent behind the plane of the page. In other cases thestereochemistry is not indicated and such structures are intended toencompass both the enantiomerically pure or purified forms of thecompound shown as well as a racemic mixture of enantiomers.

As will be readily understood, functional groups present may containprotecting groups during the course of synthesis. Protecting groups arechemical functional groups that can be selectively appended to andremoved from functionalities, such as amine, hydroxyl groups or carboxylgroups. These groups are present in a chemical compound to render suchfunctionality inert to chemical reaction conditions to which thecompound is exposed. Any of a variety of protecting groups may beemployed with the present invention. Protecting groups include thebenzyloxycarbonyl group and the tert-butyloxycarbonyl group. The term“protecting group” or “blocking group” refers to any group which whenbound to one or more hydroxyl, amino, carboxyl or other groups of thecompounds (including intermediates thereof such as the aminolactams,aminolactones, etc.) prevents reactions from occurring at these groupsand which protecting group can optionally be removed by conventionalchemical or enzymatic steps to reestablish the hydroxyl, amino, carboxylgroup for use in an IAP binding compound. The particular removableblocking group employed is not critical and preferred removable hydroxylor amine blocking groups include conventional substituents such asallyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl,t-butyl-diphenylsilyl, t-butyl carbamate, benzyl carbamate and any othergroup that can be introduced chemically onto a hydroxyl, amine, or otherfunctionality and later selectively removed either by chemical orenzymatic methods in mild conditions compatible with the nature of theproduct.

The cargo portion of the molecule may be part of the backbone or IAPbinding portion of the molecule or the cargo portion may be chemicallybonded or linked to the IAP binding portion of the molecule. Forexample, the cargo portion may be another unit of the formula (2), (3)or (5), linked by a linking group to the first unit, thereby forming adimer and may further comprise an additional cargo portion. The cargoportion may also be any of the substituents A₁, A₂, R_(1a), R_(1b), Y,R₂, or Z in molecules of formula (2), preferably the cargo portion islinked to Y, R₂, or Z in molecules of formula (2). In molecules offormula (3), or (5), the cargo portion may be any of the substituentsA₁, R_(1a), R_(1b), R₁₀, Y, M, G, or Z (includes Z_(1a) and Z_(1b)),preferably the cargo portion includes a substituent linked at Y, Z(include Z_(1a) and Z_(1b)), or R₁₀. The cargo portion of the moleculemay include but is not limited to structures, moieties, and substituentsfor imaging, therapeutics, detectable groups, probes, labels, ormarkers. The cargo portion and IAP binding portion of the molecule maybe connected by a chemical bond to the IAP binding portion including butnot limited to amide, ester, or disulfide bonds, chelation, or by alinking group such as diaminobutane or ethylene glycol and its oligomerswhere it is desirable to separate the IAP binding portion of themolecule from the cargo portion of the molecule. One or more atoms inany portion of the IAP binding cargo molecule may be a radioisotope andused for detection or treatment.

The ability to quickly assay small molecules for their effectiveness indisrupting protein-protein interactions can be used in the developmentof viable drug candidates. One aspect of the present invention comprisesan assay that can be used to test the binding affinity of a library ofIAP binding compounds for their ability to bind to the BIR domain of aninhibitor of apoptosis protein (IAP), for example the BIR3 domain ofmammalian XIAP. The assay may be based on a detectable label, which canbe a fluorogenic dye molecule that is the cargo portion of anIAP-binding cargo molecule. The detectable label may be any of thesubstituents in the molecules of formula (2), (3), or (5). For example,the detectable label may be linked to the substituent R₂, or linked tothe substituents R_(2a-c) supra in molecules having the structure offormula (2)

Similarly the detectable label may be linked to substituents like Y, Z,M, G, R₁₀, or other substituents in molecules having the structure offormula (3), or (5) and pharmaceutically acceptable salt thereof.

A detectable label included in a molecule of formula (2), (3), or (5)can be a dye such as is a fluorogenic dye whose emission is sensitive tothe environment of the dye. However, the detectable label portion of anIAP binding cargo molecule may also be an NMR-active nucleus or acontrast agent and the selective identification is performed throughnuclear magnetic resonance or magnetic resonance imaging. The detectablelabel in an IAP-binding cargo molecule may also be a radioisotope andwhere the selective identification is performed through positronemission tomography. The cargo portion of the molecule may also be usedto destroy cells through a toxic effect of the cargo portion of themolecule.

Without wishing to be bound by theory, it is believed that the moleculeof structure (2), (3), or (5) packs into the BIR domain of an IAP. Wherethe molecule of structure (2), (3), or (5) has a fluorgenic dye as acargo substituent, preferably the packing of the molecule of structure(2), (3), or (5) into the groove of the BIR of an IAP causes a largeshift in emission maximum and intensity of the dye when the environmentof the IAP binding cargo molecule changes from water to the hydrophobicpocket or binding in or near the groove of the IAP. If a molecule (e.g., the native Smac protein or a test IAP binding compound) displacesthe IAP binding cargo molecule of formula (2), (3), or (5) with thefluorgenic dye from the IAP, then emission will shift back to thespectrum observed for the IAP binding cargo molecule with thefluorogenic dye in water. Since the emission intensity is related to thebinding of a test peptide, or IAP binding compound with the IAP, theintensity can be used to estimate the equilibrium constant, K_(a), fordisplacement of the molecule of formula (2), (3), or (5) by the IAPbinding compound; K_(a) refers to an equilibrium constant forassociation that is inversely related to the dissociation K_(d)=1/K_(a).The larger the equilibrium constant K_(a), the greater affinity the IAPbinding compound has for the BIR or BIR3. This allows the most promisinginhibitors to be quickly determined, and structural information abouteffective inhibitors can be incorporated into the design of candidatesfor the next round of testing. An IAP binding cargo molecule of formula(2) having a detectable label may be complexed to an IAP and used toscreen other IAP binding compounds.

It will be understood by those of skill in the art that, although theIAP binding cargo molecule of formula (2), (3), or (5) described aboveis exemplified and preferred for practice of the invention, variouscombinations of IAP binding compounds, BIR binding domains of differentIAP's, and detectable labels may be used interchangeably to createvariations of the assay described above.

The IAP binding cargo molecule of structure (2), (3), or (5) or dimersthereof may comprise any suitable therapeutic molecule or detectablelabel, such as but not limited to a fluorophore, radioisotope, or NMRactive nucleus, such that binding of the IAP binding cargo molecule tothe BIR domain of an IAP, and preferably the BIR3 groove of XIAP, is notdetrimentally affected by the presence of the detectable label ortherapeutic in the IAP binding cargo molecule. Preferably the moleculeis cell permeable. A non-limiting example of a detectable label whichmay be coupled to the IAP binding compounds and IAP binding cargomolecules of structure (2), (3), or (5) is the fluorogenic dye6-Bromoacetyl-2-dimethylaminonaphthalene (badan) dye. Badan is afluorogenic dye whose sensitivity to environmental changes haspreviously been made use of to probe protein binding interactions.

The IAP binding cargo molecules of structure (2), (3), or (5) can beused in an assay of test compounds that can bind to the BIR domain of anIAP. These molecules may be used for example to relieve suppression ofapoptosis or to release sequestered proteins like Smac from IAPs incells. A high-throughput, cell-free assay, for compounds of structure(2), (3), or (5) may also be prepared using a fluorescently labeledpeptide like (AbuRPF—K(5-Fam)-NH₂). A wide variety of IAP bindingcompounds may be screened or assayed for their ability to bind to theBIR domain of IAPs. IAP binding molecules with greater binding abilitythan the naturally-occurring Smac, or IAP binding molecules that canrelease sequestered Smac from IAP in cells can be identified by such anassay. These compounds may be developed as therapeutic agents,pharmaceutical compositions for the modification, and preferably thepromotion, of apoptosis in treatment of diseases or pathologicalconditions in which cell proliferation plays a role. These identifiedcompounds may be used as prophylactics and can also be modified toinclude detectable labels or toxic agents. The assay may be further usedin high throughput screening of large panels of compounds generated bycombinatorial chemistry or other avenues of rational drug design. Thefluorescence assay can be used to test the binding of a library of IAPbinding cargo molecules modeled on the binding of Smac and its homologs,and preferably the binding of Smac N-terminus, to the surface pocket ofthe BIR3 region of MAP. The results of such screening make it possibleto parse the contribution of each moiety in the structure of the IAPbinding cargo molecule to the total binding of the interaction. Forexample, by comparing the binding, K_(d), of molecules (5-54) and (5-55)in Table 5 for different sized alkyl groups for R_(1b), the contributionof methylene group to the IAP binding K_(d), can be used to make furthermodifications to the molecules.

The present invention features, IAP binding compounds and methods oftheir use for binding to Inhibitor of Apoptosis Proteins (IAPs),including but not limited to XIAP, c-IAP1, c-IAP2, survivin, ML-IAP orcombinations of these. One function of IAPs is to suppress programmedcell death, whereas Smac, or IAP binding compounds of structure (2),(3), or (5) can be used to relieve that suppression. The mammalian IAPbinding protein Smac is dependent upon binding of its N-terminal fourresidues to a featured surface groove in a portion of XIAP referred toas the BIR3 domain. This binding prevents XIAP from exerting itsapoptosis-suppressing function with caspases in the cell. An IAP bindingcargo molecule, such as those of formula (2), (3), or (5), may be usedto relieve XIAP-mediated or other IAP-mediated suppression of apoptosisin mammalian cells and can optionally provide a functional group havinga detectable property or a therapeutic function to the cell. IAP bindingcargo molecule, such as those of formula (2), (3), or (5), may be usedor to release sequestered proteins like Smac from IAPs in cells.

One embodiment of the invention is a method of using versions of IAPbinding compounds of formula (2), (3), or (5) that can includeadministering to abnormal cells or tissue, which may be known tooverexpress IAP as well as other cell lines related to developmentaldisorders, cancer, autoimmune diseases, as well as neuro-degenerativedisorders such as but not limited to for example SK—OV-3 cells, HeLacells, or other cells, an amount of the IAP binding compounds or IAPbinding cargo molecules in various embodiments of formula (2), (3), or(5) that is effective to reduce, eliminate, or otherwise treat thesample of cells. An amount of the IAP binding compounds or IAP bindingcargo molecules in various embodiments of formula (2), (3), or (5) canbe administered to normal cells or tissue as a control. The amount ofcompound of formula (2), (3), or (5), combinations of these, orcombinations that include other therapeutic compounds that are effectiveto reduce the proliferation condition can be determined from changes inthe optical density of treated and control cell or tissue samples. Theadministered compound of structure (2), (3), or (5), in some embodimentsinclude those with an EC₅₀ as measured using the method described, forexample, in Example 1 for the IAP binding compounds of less than about 1micromolar, in other embodiments an EC₅₀ of less than about 0.5micromolar, and in still other embodiments the EC₅₀ for the administeredIAP binding compounds of structure (2), (3), or (5) can be less thanabout 0.06 micromolar.

The term IAP binding compound refers to a molecule that providestertiary binding or activity with the BIR-containing protein'sfunctional domain (e.g., binding motif or active site) of an IAP. TheseIAP binding compounds can be non-peptide agents such as small moleculedrugs of structure (2), (3), or (5) or that include molecules ofstructure (2), (3), or (5). Knowing the structural features and bondingof naturally-occurring IAP-binding cargo molecules such as Smac and itshomologs, it is advantageous to make IAP binding compounds that havesimilar or improved binding compared to the core IAP-binding N-terminaltetrapeptides of Smac and its homologs. One added advantages of IAPbinding cargo molecules of structure (2), (3), or (5) in variousembodiments of the invention is that compounds of this size andstructure can be prepared by large scale syntheses, they can bechemically modified to have improved solubility in aqueous solution,have improved cell permeance, and provide ease of delivery to selectedtargets in vivo.

IAP-binding cargo molecules of the invention can include amino acids aswell as molecules where the amino acids are modified to produce IAPbinding compounds by elimination, replacement or modification, of one ormore naturally occurring side chains of the genetically encoded aminoacids. Replacement can include exchange of one or more of the L aminoacids with D amino acids. Where the naturally occurring side chains ofthe amino acids are replaced, groups such as alkyl, lower alkyl, cyclic4-, 5-, 6-, to 7-membered alkyl, amide, lower alkyl amide, di(loweralkyl)amide, lower alkoxy, hydroxy, carboxy and the lower esterderivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclicscan be used. For example, proline analogs can be made in which the ringsize of the proline residue is changed from 5 members to 4, 6, or 7members or substituents are added at various positions (2, 3, 4, or 5)on the ring. For example in the structure of formula (2), thesubstituents may be an ester group R₂, or an aryloxy group Z. Cyclicgroups can be saturated or unsaturated, and if unsaturated, can bearomatic or non-aromatic. Heterocyclic groups may be used in as a sidegroup in R₂ in the molecules of formula (2), (3), or (5). Theheterocyclics can contain one or more nitrogen, oxygen, and/or sulphurheteroatoms. Examples of such heterocyclic groups include the furazanyl,fliryl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl,isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino),and triazolyl. These heterocyclic groups can be substituted orunsubstituted. Where a group is substituted, the substituent can bealkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.The IAP binding compounds may also have amino acid residues that havebeen chemically modified by phosphorylation, sulfonation, biotinylation,or the addition or removal of other moieties.

A variety of synthetic techniques known to those skilled in the art areavailable for constructing IAP binding compounds with the same orsimilar desired biological activity as the corresponding native peptidebut with more favorable activity than the peptide with respect tosolubility, stability, cell permeability, immunogenicity, and/orsusceptibility to hydrolysis or proteolysis. These IAP binding cargomolecules are synthetic compounds having a three-dimensional structure(i.e. a “peptide motif”) based upon the three-dimensional structure of aselected Smac peptide or homolog. The peptide motif provides the IAPbinding compound with the desired biological activity, i.e., binding toIAP, wherein the binding activity of the IAP binding compound is notsubstantially reduced, and is often the same as or greater than theactivity of the native peptide on which the IAP binding compound ismodeled. IAP binding cargo molecules of the present invention, can haveadditional beneficial characteristics that enhance their therapeuticapplication, such as decrease cost for synthesis, increased cellpermeability, enhanced stability to radiological elements, greateraffinity and/or avidity for target IAPs, and prolonged biologicalhalf-life.

In one class of IAP binding compounds, the backbone can include variouschemical bonds such as ester, thioester, thioamide, retroamide, reducedcarbonyl, dimethylene and ketomethylene bonds, to modify IAP binding inthe IAP binding cargo compounds.

Because caspases are cytosolic enzymes, diagnostic, imaging,prophylactic, and therapeutic IAP binding cargo molecules thatchemically bind with the IAP proteins preferably cross cell membranes.The cell membrane-permeant IAP binding cargo molecule complexes of thepresent invention are preferably those that can confer the desiredintracellular translocation and IAP binding properties to the IAPbinding cargo molecules. Preferably, these IAP binding cargo moleculesare characterized by their ability to confer transmembrane translocationand internalization of a complex IAP binding cargo molecule of structure(2), (3), or (5) when administered to the external surface of an intactcell, tissue or organ. The ability of the IAP binding cargo molecules ofthe present invention to permeate the cell and become localized withincytoplasmic and/or nuclear compartments may be demonstrated by a varietyof detection methods such as, for example, fluorescence microscopy,confocal microscopy, electron microscopy, autoradiography, orimmunohistochemistry.

Without wishing to be bound by theory, the IAP binding cargo molecule ofstructure (2), (3), or (5) can bind to IAPs in the cell and can but isnot limited to competitively displacing IAPs bound to a caspases in thecells or releasing Smac sequestered with IAPs in the cells. The IAPbinding cargo molecule chemically, physically, or by a combination ofthese, binds to an IAP protein and may displace it from a mature caspaseor Smac protein in a cell. The physical interaction between the IAPbinding portion of the IAP binding cargo molecule and an IAP protein canbe used to modify apoptosis in cells.

IAP binding molecules or cargo molecules of formula (2), (3), or (5) canhave an IAP binding portion which can, for example, displace a moleculeor polypeptide such as a mature caspase or fluorescently labeled peptide(AbuRPF—K(5-Fam)-NH₂) from the BIR domain of an IAP. Where an increasein apoptosis in cells is desirable, IAP binding molecules of formula(2), (3), or (5) having a binding constant K_(d) as measured by themethods described, for example, in Example 1 for the displacement of(AbuRPF—K(5-Fam)-NH₂) from the BIR domain of an IAP can be less thanabout 10 μM, in some embodiments K_(d) can be less than about 1 μM, andin still other embodiments K_(d) can be less than 0.1 micromolar underthe assay conditions described in the examples.

The labeled IAP-binding cargo molecule of structure (2), (3), or (5) mayinclude any suitable detectable label, including fluorophores,chromophores, fluorescent nanoparticles, and other dyes, isotopes,radioisotopes, metals, small molecules and the like. Where the label islinked or bonded to the IAP binding portion of the molecule, the labelpreferably does not interfere substantially with the cell permeance orbinding of the molecule to IAP and permits its use in diagnostic ortherapeutic applications. In selecting a label, preferably a detectableproperty of the label changes with the binding of the IAP binding cargomolecule to the BIR domain of an IAP protein. The detectable property ofthe label may change because the interaction of the label with thecellular environment changes when the molecule binds to IAP therebyenhancing or diminishing the property.

IAP-binding cargo molecules can also find utility as therapeutic agents.In one instance the binding of the IAP binding cargo molecule to IAP incells can be used to modify apoptosis in cells in need of treatment. Inanother instance, an IAP binding cargo molecule where the cargo portionis radiolabeled may be used for radiation therapy. Through the use ofthese cargo molecules, radioactive atoms may be administered to a tumor,tissue, or other population of cancer cells that overexpress IAPprotein. The IAP in the tumor becomes bonded to the IAP binding cargomolecule with the radionuclei. Similarly, IAP-binding cargo moleculesmay be designed to incorporate a dye that is active in photodynamictherapy. Other such therapeutic utilities will be apparent to thoseskilled in the art.

Cells being evaluated to detect abnormal levels of IAP in the cells maybe mixed and optionally incubated with an IAP binding cargo molecule ina fluid sample in a vessel or wells, a flowing fluid, or fluidsfollowing purification. These samples may be monitored for changes in adetectable property of the IAP binding cargo molecule. For example, flowcytometry is a method for analyzing cells labeled with a fluorescentprobe molecule on a flow cytometer. In a flow cytometer the cells passsingle-file through a focused laser beam where they emit fluorescencefrom the probe within the cell that can be detected by thephotomultiplier tubes of the cytometer. Cells with abnormal expression,high or low, of IAP may be contacted and optionally incubated with IAPbinding cargo molecules of structure (2), (3), or (5) having afluorescent probe cargo portion. The binding of the IAP binding cargomolecules to the LAP protein in the cells may be detected by the flowcytometer. The intensity of the fluorescence emission can be measured,digitized, and stored on a computer disk for analysis and comparison tothe fluorescent emission from control cells, samples of cells beingtreated, or other cell samples whose IAP expression is to be determined.

A method of screening for IAP proteins in cells with a molecule thatbinds the BIR domain in an IAP protein is provided. The method includescombining an IAP binding cargo molecule of formula (2), (3), or (5) andthe IAP proteins from cells, under conditions wherein the IAP bindingcargo molecule and IAP protein can combine. It may include the step ofincubating a sample of cells with an IAP binding cargo molecule. IAPbinding by the molecule, an indication of the presence of IAP in thecells may be determined by monitoring a detectable binding property ofthe IAP binding cargo molecule. A change in the detectable property ofthe IAP binding molecule may be used to determine the expression of IAPin the cells. Where IAP protein is over expressed in cells, the IAPbinding cargo molecule can be used to bind the IAP and relieveIAP-mediated inhibition of caspase activity in the cell. Alternatively,where an IAP protein sequesters a protein like Smac or other proteininvolved in apoptosis, the IAP binding cargo molecule of structure (2),(3), or (5) can be used to release the sequested Smac or other proteinfrom the IAP and modify apoptosis within the cells. Where desirable ornecessary in a course of treatment, other IAP binding cargo moleculesmay be administered to the cells. These additional IAP binding cargomolecules may have different binding affinity for the IAP and mayoptionally include a cargo portion that is a therapeutic agent such as aradioisotope.

The IAP-binding cargo molecules, for example those of formula (2), maybe utilized in various assays to screen for and identify compoundscapable of acting as agonists or antagonists of the IAP-caspase proteinor IAP-Smac interactions within cells. For example, IAP binding cargomolecules which can disrupt IAP-caspase interaction, antagonists of thisinteraction, are expected to be useful as pro-apoptotic drugs fortreatment of cell proliferative diseases such as cancer. Agonists ofthis interaction may be useful as anti-apoptotic drugs for treatment ofdiseases where inhibition of apoptosis is needed, e.g., degenerativediseases such as Alzheimer's disease.

A living system may include plants, animals, single and multicellularorganisms, and insects. The term mammal includes humans and all domesticand wild animals, including, without limitation, cattle, horses, swine,sheep, goats, dogs, cats, and the like.

An effective amount refers to that amount of a an IAP binding cargomolecule of formula (2), (3), or (5) of the present invention which,when administered to a sample of one or more cells including a tissue,or a living system such as an animal, preferably a mammal, in needthereof, is sufficient to effect detection of IAP in tissue or cells,prophylaxis of tissue or cells, or therapeutic treatment of IAP in thecells or tissue, preferably those in a living system. For disease-statesalleviated by the inhibition of IAP activity, the amount of a compoundof the present invention which constitutes a therapeutically effectiveamount that modifies or promotes apoptosis in one or more cellsincluding a tissue, or a subject will vary depending on the compound,the disease-state and its severity, and the mammal to be treated, butcan be determined routinely by one of ordinary skill in the art havingregard to his own knowledge and to this disclosure. A diagnosticallyeffective amount is an amount of an IAP binding cargo moleculesufficient to permit detection of IAP in cells or tissue and may forexample vary depending upon the location of the cell or tissue and thestability of the IAP binding-cargo molecule. A prophylacticallyeffective amount is an amount of an IAP binding cargo molecule thatprevents the occurrence of a disease state and may be determined forexample by prophylactic administration of IAP binding cargo molecules tocells, tissue, or test animals with controls and then exposing these toconditions known to induce abnormal cellular proliferation and thendetermining the prophylactically effective amount of the IAP bindingcargo molecule.

Treating or treatment of a disease-state in a sample of cells, a tissue,a mammal, and particularly in a human can include detecting the presenceof disease in the cells using compounds of structure (2), (3), or (5) ofthe present invention and where a disease is detected, optionallyfollowed by administration of compounds of the present invention to theone or more cells, to an animal or tissue including human subjects, tomodify and preferably promote apoptosis. The disease-state in the caseof over expression of IAP proteins in cells may be alleviated by theinhibition of an IAP-caspase interaction or an IAP-Smac interaction byadministering IAP binding cargo molecules of the present invention tothe cells thereby causing regression of the disease-state. The treatmentcan also include: preventing the disease-state from occurring in amammal. For example, in a mammal that is predisposed to a disease-statecharacterized by inhibition of apoptosis, but the mammal has not yetbeen diagnosed as having the disease; an effective amount of the IAPbinding compounds of the present invention may be administered to cellsor the patient to inhibit the disease-state or arrest its development.

In one embodiment, molecules of structure (2), (3), of (5) can beadministered as a composition to provide systemic distribution of theIAP binding molecules such as by oral, buccal or parenteraladministration in the mammal or human. The administration can beincluded in a method of treating mammals, especially humans, sufferingfrom a proliferation disorder or that are at risk of a proliferationdisorder. “At risk” refers to mammals like persons whose genotype,family history, or other risk factors indicates a greater than normallikelihood that the person will suffer from a proliferation disorder ifleft untreated.

The expression of IAP in cells can be detected in patients without theneed for surgery. Accordingly, the present invention encompassescompounds and methods for detecting intracellular biochemical activitiesin living systems such as, whole animals, tissues, or cells, byadministering IAP binding cargo molecules of this invention whichtranslocate into cells, and which are detectable in living cells atdistances removed from the cells by the presence of intervening tissue.The methods and compositions can be used for identifying cells or tissuehaving abnormal expression of IAP in a combination of one or more cellsor tissues; and administering an effective amount of an IAP-bindingcargo molecule to bind with the IAP in the sample cells and modify theactivity of the IAP in the cells or tissue. Examples of tissues to whichthe methods and compositions of the present invention can be appliedinclude, for example, cancer cells, in particular, central nervoussystem tumors, breast cancer, liver cancer, lung, head and neck cancer,lymphomas, leukemias, multiple myeloma, bladder cancer, ovarian cancer,prostate cancer, renal tumors, sarcomas, colon and othergastrointestinal cancers, metastases, and melanomas. Other examples ofdiseases, conditions or disorders where modification of apoptosis orabnormal IAP activity are involved and to which the methods andcompositions of the present invention can be applied include, but arenot limited to infection, inflammation, neurodegenerative diseases suchas Alzheimer disease and Parkinson's disease. A proliferation disordermay include a disorder in which IAP activity inhibits apoptosis in cellsreceiving an apoptotic stimulus.

Apoptosis may be promoted in a sample of cells by administering to thecells an amount of an IAP-binding cargo molecule effective to stimulateapoptosis in the cells. The cells may be cultured cells, cells fromwithin a tissue, and the tissue preferably is located within a livingorganism, preferably an animal, more preferably a mammal, and mostpreferably a human. These latter embodiments are carried out byformulating the IAP-binding cargo molecules of the invention in atherapeutically effective amount as a pharmaceutical preparation foradministration to a mammalian subject. Such a pharmaceutical preparationconstitutes another aspect of the present invention.

The ability of a pharmaceutical agent to simulate or inhibit apoptosismay be tested in a cell-free activity assay of downstream targets ofIAP. In the absence of an IAP-binding cargo molecule, IAP itself can forexample interact with Smac or inhibit the activity of caspases, therebyarresting apoptosis. Such assays include, but are not limited to, directcaspase-9 activity assays and caspase activation assays (cleavage ofprocaspases). In these assays, an IAP-binding cargo molecule of theinvention, having a pre-determined level of activity in such assays, isused as a positive control and, optionally, a corresponding moleculeknown not to be active in the assay is used as a negative control.Assays can be conducted using these controls, and the cells undergoingthe treatment evaluated on relief of inhibition of Smac or caspaseactivity by IAP in the presence of the IAP binding cargo molecule ofstructure (2), (3), or (5). Cells that undergo apoptosis can bedifferentiated from normal cells by distinct morphological changes or bymolecular markers, such as cleavage of chromosomes into nucleosomeladders (detected by nuclear DNA staining).

Pharmaceutically active or biologically active IAP binding cargomolecules of the invention are those that bind IAP (inhibitor ofapoptosis protein), specifically the BIR domain of IAP, morespecifically the BIR3 binding groove of XIAP. This biological activitymay be measured with respect to any IAP, including but not limited toXIAP, c-IAP1, c-IAP2, survivin, ML-IAP, and DIAP.

Various aspects of the present invention will be illustrated withreference to the following non-limiting examples.

Example 1

Binding constants (K_(d)) were measured using fluorescence polarizationas described by Zaneta Nikolovska-Coleska, Renxiao Wang, Xueliang Fang,Hongguang Pan, York Tomita, Peng Li, Peter P. Roller, KrzysztofKrajewski, Naoyuki Saito, Jeanne Stuckey and Shaomeng Wang, in“Development and Optimization of a Binding Assay for the XIAP BIR3Domain Using Fluorescence Polarization”, Analytical Biochemistry 2004,332, 261-273). Briefly, test IAP binding compounds at variousconcentrations for binding measurements were mixed with 5 nMfluorescently labeled peptide (AbuRPF—K(5-Fam)-NH₂) and 40 nM ofXIAP-BIR3 for 15 minutes at room temperature in 100 μL of 0.1M PotassiumPhosphate buffer, pH 7.5 containing 100 μg/ml bovine γ-globulin.Following incubation, the polarization values (mP) were measured on aVictor²V using a 485 nm excitation filter and a 520 nm emission filter.IC₅₀ values were determined from the plot using nonlinear least-squaresanalysis using GraphPad Prism. The K_(d) values of competitiveinhibitors were calculated using the equation described by by ZanetaNikolovska-Coleska et al. based upon the measured IC₅₀ values, the K_(d)value of the probe and XIAP BIR3 complex, and the concentrations of theprotein and probe in the competition assay. In various examples of IAPbinding or IAP binding cargo molecules ranges for K_(d) values: Group A:K_(d)<0.1 μM; Group B: K_(d)=0.1−1 μM; Group C: K_(d)=1−10 μM; Group D:K_(d)>10 μM.

Example 2

This example illustrates use of compounds of embodiments of the presentinvention that can be used in a method of treating cells. The methodusing these IAP binding compounds can include administering to abnormalcells, which may be known to overexpress IAP as well as other cell linesrelated to developmental disorders, cancer, autoimmune diseases, as wellas neuro-degenerative disorders such as but not limited to for exampleSK—OV-3 cells, HeLa cells or other cells, an amount of the IAP bindingcompounds or IAP binding cargo molecules in various embodiments offormula (2), (3), or (5) that is effective to reduce, eliminate, orotherwise treat the sample of cells.

The MTT assay is an example of an assay that has been used for measuringcell growth as previously described (Hansen, M. B., Nielsen, S. E., andBerg, K. J. Immunol. Methods 1989, 119, 203-210) and incorporated hereinby reference in its entirety. Briefly, SK—OV-3 cells were seeded in96-well plates in McCoy's medium containing 10% fetal bovine serumalbumin (20,000 per well) and incubated overnight at 37° C. Next day,test compounds were added at various concentrations typically from about10 to about 0.0001 μM and the plates were incubated at 37° C. for anadditional 72 hrs. This incubation time was optimal for measuringinhibitory effects of different analogs. 50 microliters of 5 mg/mL MTTreagent to each well was added and the plates were incubated at 37° C.for 3 hours. At the end of incubation period, 50 microliters of DMSO wasadded to each well to dissolve cells and the optical density (OD) of thewells was measured with a microplate reader (Victor²1420, Wallac,Finland) at 535 nm. Cell survival (CS) was calculated by the followingequation:CS=(OD treated well/mean OD control wells)×100%

The EC₅₀, defined as the drug concentration that results in 50% CS, wasderived by calculating the point where the dose-response curve crossesthe 50% CS point using GraphPad Prism.

Representative results for IAP binding compounds are:

TABLE 1 EC₅₀, μM Entry A₁ R_(1b) Y Z R₁₂ R₁₄₋₁₇ R₁₁ (±sd) 1-1 Me Me tBuH MeO(CH₂CH₂O)₂CH₂CH₂ 6-F H 0.092 ± 0.077 (R₁₆) 1-2 Me Me cHex HMeO(CH₂CH₂O)₂CH₂CH₂ 6-F H 0.060 ± 0.026 (R₁₆) 1-3 Me Me cHex H H 6-F H0.515 ± 0.093 (R₁₆)

Example 3

This example illustrates the preparation of pyrrolidine derivatives ofTable 2. The examples include molecules of formula (E3) that can includeheteroalkynyl substituents.

The Preparation of2S-Amino-N-[2-methyl-1S-(4S-phenoxy-2S-phenoxymethyl-pyrrolidine-1-carbonyl)-propyl]-propionamidehydrochloride (7) A.(4S)-Phenoxy-(2S)-phenoxymethyl-pyrrolidine-1-carboxylic acid tert-butylester (2)

To a solution of alcohol 1 (0.53 g, 1.8 mmol) in anhydrous DCM (10 mL)was added phenol (0.21 g, 2.3 mmol), Ph₃P (0.52 g, 2.0 mmol), and1,1′-(azodicarbonyl)-dipiperidine (ADDP, 0.50 g, 1.9 mmol) in sequentialorder. After 2 hr at ambient temperature, the heterogeneous reactionmixture was filtered. The white solid was washed with DCM and theclarified filtrate was washed with 2M NaOH, water, brine, dried withanhydrous Na₂SO₄, filtered and concentrated. The crude aryl ether waspurified by flash silica gel chromatography (3:1 hexane/EtOAc) to afford0.36 g (54%) of 2 as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ7.29-7.24 (m, 4H), 6.94-6.82 (m, 6H), 4.91 (t, J=5.1 Hz, 1H), 4.37-4.28(m, 2H), 4.12-4.05 (m, 1H), 3.79-3.64 (m, 2H), 2.48 (app d, J=13.8 Hz,1H), 2.32-2.25 (m, 1H), 1.48 (s, 9H) ppm.

B. (4S)-Phenoxy-(2S)-phenoxymethyl-pyrrolidine (3)

Trifluoroacetic acid (2 mL) was added at ambient temperature to asolution of 2 (0.36 g, 0.98 mmol) in DCM (10 mL). After 1.5 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated. The crudepyrrolidine was purified by flash silica gel chromatography (10%MeOH/DCM) to afford 0.23 g (88%) of 3 as a brown-orange oil. ¹H NMR(CDCl₃, 300 MHz) δ 7.32-7.26 (m, 4H), 6.98-6.87 (m, 6H), 4.90-4.87 (m,1H), 4.07-4.01 (m, 2H), 3.63-3.57 (m, 1H), 3.35 (app d, J=12.3 Hz, 1H),3.17 (app q, J=5.4 Hz, 1H), 2.77 (br s, 1H), 2.45-2.35 (m, 1H),1.93-1.86 (m, 1H) ppm.

C.[2-Methyl-1-(4S-phenoxy-2S-phenoxymethyl-pyrrolidine-1-carbonyl)-propyl]-carbamicacid tert-butyl ester (4)

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium PF₆ (HATU, 0.38g, 1.0 mmol) was added to a solution of N-Boc-Val (0.28 g, 1.3 mmol) inanhydrous NMP (5 mL) at ambient temperature. N-Methylmorpholine (0.1 mL)was added to reaction mixture. After 10 min, pyrrolidine 3 (0.23 g, 0.86mmol) in NMP (5 mL) was added. After 2 h, the reaction mixture wasdiluted with EtOAc and washed with dilute aqueous NaHCO₃, 1N HCl, water,and brine. The organic phase was dried over anhydrous Na₂SO₄, filteredand concentrated. The crude amide was purified by flash silica gelchromatography (3:1 hexane/EtOAc) to afford 0.36 g (90%) of 4 as acolorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.34-7.26 (m, 4H), 7.03-6.82(m, 6H), 5.30 (d, J=9.3 Hz, 1H), 5.01-4.99 (m, 1H), 4.69-4.65 (m, 1H),4.38-4.34 (m, 1H), 4.27-4.07 (m, 3H), 3.83-3.78 (m, 1H), 2.53-2.48 (m,1H), 2.38-2.31 (m, 1H), 2.00-1.94 (m, 1H), 1.48 (s, 9H), 0.97 (d, J=7.2Hz, 3H), 0.93 (d, J=7.2 Hz, 3H) ppm.

D.2-Amino-3-methyl-1-(4S-phenoxy-2S-phenoxymethyl-pyrrolidin-1-yl)-butan-1-one(5)

Trifluoroacetic acid (2 mL) was added at ambient temperature to asolution of 4 (0.36 g, 0.79 mmol) in DCM (10 mL). After 1.5 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude aminewas purified by flash silica gel chromatography (5% MeOH/DCM) to afford0.27 g (96%) of 5 as a light yellow oil. ¹H NMR (CDCl₃, 300 MHz) δ7.32-7.01 (m, 4H), 7.01-6.82 (m, 6H), 4.99 (m, 1H), 4.67-4.48 (m, 1H),4.38-4.27 (m, 1H), 4.16-4.05 (m, 1H), 3.99 (app q, J=5.1 Hz, 1H),3.78-3.72 (m, 1H), 2.50-2.28 (m, 2H), 1.86 (br s, 2H), 0.97 (d, J=7.2Hz, 3H), 0.91 (d, J=7.2 Hz, 3H) ppm.

E.{1S-[2-Methyl-1S-(4S-phenoxy-2S-phenoxymethyl-pyrrolidine-1-carbonyl)-propylcarbamoyl]-ethyl}-carbamicacid tert-butyl ester (6)

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium PF₆ (HATU, 0.23g, 0.61 mmol) was added to a solution of N-Boc-Ala (0.14 g, 0.74 mmol)in anhydrous NMP (3 mL) at ambient temperature. N-Methylmorpholine (0.1mL) was added to reaction mixture. After 10 min, pyrrolidine 5 (0.17 g,0.48 mmol) in NMP (5 mL) was added. After 16 h, the reaction mixture wasdiluted with EtOAc and washed with dilute aqueous NaHCO₃, 1N HCl, water,and brine. The organic phase was dried over anhydrous Na₂SO₄, filteredand concentrated. The crude amide was purified by flash silica gelchromatography (1:1 hexane/EtOAc) to afford 0.23 g (92%) of 6 as acolorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.33-7.23 (m, 4H), 7.02-6.87(m, 6H), 5.02-4.99 (m, 2H), 4.68-4.48 (m, 2H), 4.33 (dd, J=3.9, 9.3 Hz,1H), 4.23-4.09 (m, 4H), 3.83-3.78 (m, 1H), 2.52-2.47 (m, 1H), 2.38-2.28(m, 1H), 2.08-1.99 (m, 1H), 1.80 (br s, 1H), 1.45 (s, 9H), 1.36 (d,J=6.9 Hz, 3H), 0.95 (d, J=6.9 Hz, 3H), 0.90 (d, J=7.0 Hz, 3H) ppm.

F.2S-Amino-N-[2-methyl-1S-(4S-phenoxy-2S-phenoxymethyl-pyrrolidine-1-carbonyl)-propyl]-propionamidehydrochloride (7)

Trifluoroacetic acid (2 mL) was added at ambient temperature to asolution of 6 (0.23 g, 0.44 mmol) in DCM (10 mL). After 1.5 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude aminewas dissolved in diethyl ether then treated with HCl(g). The crude saltwas triturated with diethyl ether and hexane to afford 0.13 g (65%) of 7as a white solid. ¹H NMR, free base (CDCl₃, 300 MHz) δ 7.89-7.87 (m,1H), 7.32-7.24 (m, 4H), 7.01-6.79 (m, 5H), 5.01-4.98 (m, 1H), 4.68-4.62(m, 1H), 4.51-4.43 (m, 1H), 4.36-4.26 (m, 2H), 4.18-4.10 (m, 1H),3.84-3.78 (m, 1H), 2.51-2.46 (m, 1H), 2.35-2.28 (m, 1H), 2.09-2.02 (m,1H), 1.38-1.25 (m, 4H), 0.96-0.92 (m, 6H) ppm. Mass spectra, m/z=440[(M+H)⁺].

TABLE 2 R₃₋₅, MS K_(D) Entry A₁ R_(1b) Y Z R′₃₋₅, R₄ (M + H)⁺ Range 2-1H Me iPr (S)—OPh H 440.3 B 2-2 Me Me iPr (S)—OPh H 454.3 B 2-3 H Me iPrH H 348.3 B 2-4 H Me iPr H 2,3-(CH₂)₄ 402.3 B 2-5 Me Me iPr H 2-Ph 438.3B 2-6 H Me iPr H 2-Ph 424.2 B 2-7 H Me —CH(Me)OCH₂C≡CH (S)—OPh H 480.2 A

Example 4

This example illustrates the preparation of substituted pyrrolidines ofTable 3.

The Preparation oftrans-2S-Amino-N-(1S-{2S-[3-(2-ethyl-phenoxy)-propenyl]-4S-phenoxy-pyrrolidine-1-carbonyl}-2-methyl-propyl)-propionamide(14) A.trans-2S-[3-(2-Ethyl-phenoxy)-propenyl]-4S-phenoxy-pyrrolidine-1-carboxylicacid tert-butyl ester (9)

To a solution of alcohol 8 (0.3 g, 0.94 mmol) in DCM (6 mL) was added2-ethylphenol (0.14 g, 1.16 mmol) and Ph₃P (0.27 g, 1.03 mmol). Thesolution was cooled to 0° C. and ADDP (0.28 g, 1.13 mmol) was added inone portion. After 10 min, the reaction mixture was allowed to warm toambient temperature and stirring was continued for 16 h. The whiteprecipitate was removed by filtration and washed with DCM. The clarifiedfiltrate was washed successively with 1M NaOH, water, and brine. Theorganic phase was dried with anhydrous Na₂SO₄, filtered, andconcentrated. The crude ether was purified by flash silica gelchromatography (3:1 hexane/EtOAc) to afford 0.18 g (45%) of 9 as acolorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.30-7.26 (m, 2H); 7.15 (m,2H); 7.00-6.79 (m, 4H); 6.01-5.98 (m, 1H), 5.84 (m, 1H); 4.92 (br s,1H); 4.53-4.43 (m, 3H); 3.76 (m, 2H); 2.65 (q, 2H); 2.39 (m, 1H); 2.16(d, 1H); 1.46 (s, 9H), 1.21 (t, 3H) ppm.

B. trans-2S-[3-(2-Ethyl-phenoxy)-propenyl]-4S-phenoxy-pyrrolidine (10)

Trifluoroacetic acid (2 mL) was added at ambient temperature to asolution of 9 (0.18 g, 0.43 mmol) in DCM (10 mL). After 1 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous MgSO₄, filtered, and concentrated. The crude amine(10) was used without further purification (0.13 g obtained). ¹H NMR(CDCl₃, 300 MHz) δ 7.31-7.25 (m, 3H); 7.17-7.11 (m, 2H); 6.97-6.80 (m,4H); 5.97-5.88 (m, 2H); 4.86 (m, 1H); 4.54-4.50 (m, 2H); 3.70 (q, 1H);3.34 (d, 1H); 3.07 (d of d, 1H); 2.66 (q, 2H); 2.48-2.41 (m, 1H);1.82-1.75 (m, 2H); 1.20 (t, 3H) ppm.

C.trans-(1S-{2S-[3-(2-Ethyl-phenoxy)-propenyl]-4S-phenoxy-pyrrolidine-1-carbonyl}-2-methyl-propyl)-carbamicacid tert-butyl ester (11)

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium PF₆ (HATU, 0.23g, 0.62 mmol) was added to a solution of N-Boc-Val (0.18 g, 0.82 mmol)in anhydrous NMP (3 mL) at ambient temperature. N-Methylmorpholine (0.12mL) was added to reaction mixture. After 10 min, amine 10 (0.13 g, 0.41mmol) in NMP (3 mL) was added. After 16 h, the reaction mixture wasdiluted with EtOAc and washed with dilute aqueous NaHCO₃, 1N HCl, water,and brine. The organic phase was dried over anhydrous Na₂SO₄, filteredand concentrated. The crude amide was purified by flash silica gelchromatography (3:2 hexane/EtOAc) to afford 0.20 g (92%, 2 steps) of 11as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.24 (m, 2H);7.15-7.06 (m, 2H); 6.99 (t, 1H); 6.89-6.77 (m, 4H); 5.96-5.91 (m, 2H);5.23-5.20 (m, 1H); 5.01-4.86 (m, 2H); 4.53 (m, 2H); 4.21-4.09 (m, 2H);4.09-3.97 (m, 1H); 2.68 (q, 2H); 2.36-2.32 (m, 1H); 2.18 (d, 1H);1.97-1.91 (m, 1H); 1.44 (s, 9H); 1.29-1.18 (m, 6H); 0.99-0.81 (m, 6H)ppm.

D.trans-2S-Amino-1-{2S-[3-(2-ethyl-phenoxy)-propenyl]-4S-phenoxy-pyrrolidin-1-yl}-3-methyl-butan-1-one(12)

Trifluoroacetic acid (2 mL) was added at ambient temperature to asolution of 11 (0.20 g, 0.39 mmol) in DCM (10 mL). After 1 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous MgSO₄, filtered, and concentrated. The crude amine(12) was used without further purification (0.14 g obtained). ¹H NMR(CDCl₃, 300 MHz) δ 7.28-7.26 (m, 2H); 7.15 (m, 2H); 6.99 (m, 1H);6.89-6.76 (m, 4H); 6.10-5.86 (m, 2H); 5.01-4.95 (m, 2H); 4.56 (m, 3H);3.92-3.79 (m, 1H); 3.31 (m, 1H); 2.66 (q, 2H); 2.32-2.16 (m, 1H); 1.68(m, 4H); 1.28-1.19 (m, 3H); 0.97-0.81 (m, 6H) ppm.

E.trans-[1S-(1S-{2S-[3-(2-Ethyl-phenoxy)-propenyl]-4S-phenoxy-pyrrolidine-1-carbonyl}-2-methyl-propylcarbamoyl)-ethyl]-carbamicacid tert-butyl ester (13)

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium PF₆ (HATU, 0.19g, 0.51 mmol) was added to a solution of N-Boc-Ala (0.13 g, 0.67 mmol)in anhydrous NMP (3 mL) at ambient temperature. N-Methylmorpholine (0.1mL) was added to reaction mixture. After 10 min, amine 12 (0.14 g, 0.34mmol) in NMP (3 mL) was added. After 72 h, the reaction mixture wasdiluted with EtOAc and washed with dilute aqueous NaHCO₃, 1N HCl, water,and brine. The organic phase was dried over anhydrous Na₂SO₄, filteredand concentrated. The crude amide was purified by flash silica gelchromatography (1:1 hexane/EtOAc) to afford 0.11 g (49%, 2 steps) of 13as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.31-7.22 (m, 2H);7.15-6.95 (m, 3H); 6.88-6.76 (m, 4H); 5.96-5.87 (m, 2H); 5.19 (br, 1H);5.00 (m, 1H); 4.86 (t, 1H); 4.51-4.49 (m, 3H); 4.17-4.10 (m, 2H);3.89-3.80 (m, 1H); 2.64 (q, 2H); 2.34-2.29 (m, 1H); 2.21-2.14 (m, 1H);2.04-1.98 (m, 1H); 1.44 s, 9H); 1.35-1.23 (m, 4H); 1.17 (t, 3H);0.97-0.87 (m, 6H) ppm.

F. trans-2S-Amino-N-(1S-{2S-[3-(2-ethyl-phenoxy)-propenyl]-4S-phenoxy-pyrrolidine-1-carbonyl}-2-methyl-propyl)-propionamide(14)

Trifluoroacetic acid (2 mL) was added at ambient temperature to asolution of 13 (0.11 g, 0.18 mmol) in DCM (10 mL). After 1 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated to afford 0.068g (76%) of 14 as a white solid. ¹H NMR (CDCl₃, 300 MHz) δ 7.86 (br, 1H);7.31-7.22 (m, 2H); 7.15-7.05 (m, 2H); 6.98 (t, 1H); 6.88-6.76 (m, 4H);5.97-5.86 (m, 2H); 5.00-4.98 (m, 1H); 4.88-4.83 (m, 1H); 4.57-4.51 (m,2H); 4.44 (t, 1H); 4.21 (d of d, 1H); 3.89-3.82 (m, 1H); 2.64 (q, 2H);2.33-2.29 (m, 1H); 2.17 (d, 1H); 2.04-2.00 (m, 1H); 1.36-1.25 (m, 6H);1.17 (t, 3H); 0.99-0.88 (m, 6H) ppm.

TABLE 3 R_(3-5,) MS K_(D) Entry A₁ R_(1b) Y Z R′₃₋₅, R₄ (M + H)⁺ Range3-8 H Me iPr OPh H 466.2 B 3-9 H Me iPr OPh 2-Br 544.0 B 3-10 H Me iPrOPh 2,3-di-Cl 534.1 B 3-11 H Me iPr OPh 2,4-di-Cl 534.1 B 3-12 H Me iPrOPh 2-Et 494.4 B 3-13 Me Me iPr OPh 2,3-di-Cl 548.2 B 3-14 H Me iPr OPh3-Br 544.1 B 3-15 H Me iPr OPh 2-iPrO 524.3 B 3-16 Boc Me iPr OPh3,4-(CH₂)₄ 620.3 D 3-17 H Me iPr OPh 3,4-(CH₂)₄ 520.3 C 3-18 H Me iPrOPh 2-MeO 496.3 B 3-19 H H iPr OPh 2-MeO 482.3 D 3-20 H Me iPr OPh 2-Ph542.3 B 3-21 H Me iPr OPh 2- 534.3 C cyclopentyl 3-22 H Me iPr OPh2,6-di-Me 494.3 A

Example 5

This example illustrates the preparation of pyrrolidine derivatives ofTable 4.

The Preparation of2S-Amino-N-(1S-{2S-[3-(2-ethyl-phenoxy)-propyl]-4S-phenoxy-pyrrolidine-1-carbonyl}-2-methyl-propyl)-propionamide(21) A. 2S-(3-Hydroxy-propyl)-4S-phenoxy-pyrrolidine-1-carboxylic acidtert-butyl ester (15)

[Re: SRR-006-062] Palladium-on-carbon (10%, 0.3 g) was added to asolution of alcohol 8 (3 g, 9.4 mmol) in EtOAc (30 mL). The reactionmixture was placed on a Parr shaker and pressurized to 45 PSI H₂ (g).The heterogeneous mixture was shaken for 24 h at ambient temperature.The catalyst was removed via filtration through a 0.45 μm PVDF filterdisc (Millipore Millex-HV®) and the clarified filtrate was concentratedin vacuo to afford 2.9 g (97%) of 15 which was used without furtherpurification. ¹H NMR (CDCl₃, 300 MHz) δ 7.29 (t, 2H); 6.96 (t, 1H); 6.85(d, 2H); 4.88 (br s, 1H); 4.08-3.74 (m, 2H); 3.67-3.65 (m, 2H); 3.55 (d,1H); 2.27-2.23 (m, 2H); 2.05-1.90 (m, 2H); 1.76-1.65 (m, 1H); 1.55 (q,2H); 1.47 (s, 9H) ppm.

B. 2S-[3-(2-Ethyl-phenoxy)-propyl]-4S-phenoxy-pyrrolidine-1-carboxylicacid tert-butyl ester (16)

To a solution of alcohol 15 (0.61 g, 1.91 mmol) in DCM (10 mL) was added2-ethylphenol (0.28 g, 2.33 mmol) and Ph₃P (0.55 g, 2.09 mmol). Thesolution was cooled to 0° C. and ADDP (0.58 g, 2.29 mmol) was added inone portion. After 10 min, the reaction mixture was allowed to warm toambient temperature and stirring was continued for 16 h. The whiteprecipitate was removed by filtration and washed with DCM. The clarifiedfiltrate was washed successively with 1M NaOH, water, and brine. Theorganic phase was dried with anhydrous Na₂SO₄, filtered, andconcentrated. The crude ether was purified by flash silica gelchromatography (3:1 hexane/EtOAc) to afford 0.46 g of 16 as a colorlessoil. ¹H NMR (CDCl₃, 300 MHz) δ 7.28 (t, 2H); 7.13 (t, 2H); 6.96 (t, 1H);6.89-6.84 (m, 3H); 6.78 (d, 1H); 4.91-4.87 (m, 1H); 3.96 (br, 4H); 3.56(d, 1H); 2.63 (q, 2H); 2.28-2.26 (m, 1H); 2.11-2.06 (m, 2H); 1.86-1.77(m, 3H); 1.46 (s, 9H); 1.17 (t, 3H) ppm.

C. 2S-[3-(2-Ethyl-phenoxy)-propyl]-4S-phenoxy-pyrrolidine (17)

Trifluoroacetic acid (2 mL) was added at ambient temperature to asolution of 16 (0.46 g, 1.08 mmol) in DCM (10 mL). After 1 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated to afford 0.30 g(85%) of 17 as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.26 (m,3H); 7.16-7.12 (m, 2H); 6.986.78 (m, 4H); 4.86 (s, 1H); 3.99-3.97 (m,2H); 3.35 (d, 1H); 3.23 (m, 1H); 3.08 (m, 1H); 2.74-2.60 (m, 3H); 2.42(m, 2H); 1.90-1.85 (m, 3H); 1.67-1.61 (m, 1H); 1.16 (t, 3H) ppm.

D.(1s-{2S-[3-(2-Ethyl-phenoxy)-propyl]-4S-phenoxy-pyrrolidine-1-carbonyl}-2-methyl-propyl)-carbamicacid tert-butyl ester (18)

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium PF₆ (HATU, 0.39g, 1.03 mmol) was added to a solution of N-Boc-Val (0.30 g, 1.38 mmol)in anhydrous NMP (3 mL) at ambient temperature. N-Methylmorpholine (0.2mL) was added to reaction mixture. After 10 min, amine 17 (0.22 g, 0.67mmol) in NMP (3 mL) was added. After 16 h, the reaction mixture wasdiluted with EtOAc and washed with dilute aqueous NaHCO₃, 1N HCl, water,and brine. The organic phase was dried over anhydrous Na₂SO₄, filteredand concentrated. The crude amide was purified by flash silica gelchromatography (3:1 hexane/EtOAc) to afford 0.30 g (83%) of 18 as acolorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.33-7.26 (m, 2H); 7.12 (t,2H); 6.99 (t, 1H); 6.89-6.77 (m, 4H); 5.28-5.24 (m, 1H); 5.00-4.98 (m,1H); 4.39-4.36 (m, 1H); 4.22-4.17 (m, 2H); 4.02-3.94 (m, 2H); 3.72 (d ofd, 1H); 2.61 (q, 2H); 2.30-2.24 (m, 1H); 2.14-2.07 (, 1H); 1.98-1.89 (m,1H); 1.82-1.74 (m, 4H); 1.44 (s, 9H); 1.18-1.13 (m, 3H); 0.99-0.93 (m,6H) ppm.

E.2S-Amino-1-{2S-[3-(2-ethyl-phenoxy)-propyl]-4S-phenoxy-pyrrolidin-1-yl}-3-methyl-butan-1-one(19)

Trifluoroacetic acid (2 mL) was added at ambient temperature to asolution of 18 (0.30 g, 0.57 mmol) in DCM (10 mL). After 1 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated to afford 0.23 gof 19 as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.33-7.28 (m, 2H);7.17-7.12 (m, 2H); 6.99 (t, 1H); 6.89-6.83 (m, 3H); 6.78 (d, 1H); 4.98(br, 2H); 4.39 (br, 1H), 4.07-3.92 (m, 4H); 3.75-3.67 (m, 1H); 2.61 (q,2H); 2.31-2.24 (m, 2H); 2.13-2.08 (m, 2H); 1.94-1.75 (m, 6H); 1.16 (t,3H); 1.01-0.85 (m, 6H) ppm.

F.[1S-(1S-{2S-[3-(2-Ethyl-phenoxy)-propyl]-4S-phenoxy-pyrrolidine-1-carbonyl}-2-methyl-propylcarbamoyl)-ethyl]-carbamicacid tert-butyl ester (20)

O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium PF₆ (HATU, 0.11g, 0.27 mmol) was added to a solution of N-Boc-Ala (0.07 g, 0.37 mmol)in anhydrous NMP (3 mL) at ambient temperature. N-Methylmorpholine (0.05mL) was added to reaction mixture. After 10 min, amine 19 (0.078 g, 0.18mmol) in NMP (1 mL) was added. After 16 h, the reaction mixture wasdiluted with EtOAc and washed with dilute aqueous NaHCO₃, 1N HCl, water,and brine. The organic phase was dried over anhydrous Na₂SO₄, filteredand concentrated. The crude amide was purified by flash silica gelchromatography (1:1 hexane/EtOAc) to afford 0.038 g of 20 as a colorlessoil. ¹H NMR (CDCl₃, 300 MHz) δ 7.33-7.26 (m, 2H); 7.12 (t, 2H); 6.99 (t,1H); 6.88-6.76 (m, 4H); 5.08-5.00 (m, 1H); 4.98-4.96 (m, 1H); 4.52-4.47(m, 1H); 4.374.34 (m, 1H); 4.21-4.16 (m, 2H); 3.98-3.92 (m, 2H); 3.75(d, 1H); 2.61 (q, 2H); 2.32-2.22 (m, 1H); 2.14-1.99 (m, 4H); 1.86-1.77(m, 3H); 1.44-1.41 (m, 8H); 1.36-1.13 (m, 4H); 1.16 (t, 3H); 0.96-0.88(m, 6H) ppm.

G.2S-Amino-N-(1S-{2S-[3-(2-ethyl-phenoxy)-propyl]-4S-phenoxy-pyrrolidine-1-carbonyl}-2-methyl-propyl)-propionamide(21)

Trifluoroacetic acid (1 mL) was added at ambient temperature to asolution of 20 (0.038 g, 0.06 mmol) in DCM (5 mL). After 1 h, thesolution was concentrated to dryness and the crude product was dissolvedin EtOAc. The organic solution was washed with aqueous NaHCO₃, brine,dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude aminewas dissolved in diethyl ether then treated with HCl(g). The crude saltwas triturated with diethyl ether and hexane to afford 0.022 g of 21 asa white solid. ¹H NMR (CDCl₃, 300 MHz) δ 7.87 (br, 1H); 7.33-7.25 (m,2H); 7.12 (t, 2H); 6.96 (t, 1H), 6.89-6.77 (m, 4H); 4.98-4.96 (m, 1H);4.45 (t, 1H); 4.37-4.25 (m, 2H); 4.00-3.94 (m, 2H); 3.77 (d, 1H); 2.61(q, 2H); 2.32-2.21 (m, 1H); 2.14-2.04 (m, 3H); 1.61 (br, 2H); 1.35-1.25(m, 4H); 1.16 (t, 3H); 0.97-0.89 (m, 6H) ppm.

TABLE 4 R_(3-5,) MS K_(D) Entry A₁ R_(1b) Y Z R′₃₋₅, R₄ (M + H)⁺ Range4-23 H Me iPr OPh 2-Br 524.3 B 4-24 H Me iPr OPh 2-Ph 543.7 B 4-25 H MeiPr OPh 2-Et 552.4 B 4-26 Me Me iPr OPh 2-Et 510.3 B 4-27 H H iPr OPh2-Et 482.3 D 4-28 Me Me tBu OPh 2-Et 524.2 B 4-29 H Me iPr OPh 2-iPrO526.3 B 4-30 H Me iPr OPh 2-iPr 510.3 B 4-31 H Me iPr OPh 2,6-di- 496.2B Me 4-32 H Me iPr H 2-Ph 452.3 B 4-33 H H iPr H 2-Ph 438.3 D 4-34 H MeiPr H 2-Et 404.3 C 4-35 H Me iPr H 2-iPrO 434.3 C 4-36 H (R)—Me iPr H2-Ph 452.3 D 4-37 H Me iPr H H 376.3 C 4-38 H Me iPr H 2,6-di- 404.3 DMe

Example 6

This example illustrated pyrrolidine derivatives of Table 5.

The Preparation of2S-Amino-N-[1S-(2S-benzofuran-3-ylmethyl-pyrrolidine-1-carbonyl)-2-methyl-propyl]-propionamide(29) A. trans-2S-[3-(2-Iodo-phenoxy)-propenyl]-pyrrolidine-1-carboxylicacid tert-butyl ester (23)

To a solution of alcohol 22 (10.4 g, 0.04 mol) in DCM (300 mL) was added2-iodophenol (12.1 g, 0.05 mol) and Ph₃P (14.4 g, 0.05 mol). At ambienttemperature, ADDP (13.8 g, 0.05 mol) was added in one portion. After 16h, the white precipitate was removed by filtration and washed with DCM.The clarified filtrate was washed successively with 1M NaOH, water, andbrine. The organic phase was dried with anhydrous Na₂SO₄, and thenfiltered through a short column of silica gel. The product was elutedwith DCM and then 1:1 hexane/EtOAc to afford 15.5 g (79%) of 23 as acolorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.76 (dd, J=7.6, 1.1 Hz, 1H),7.27 (app t, J=7.6, 1.1 Hz, 1H), 6.78 (dd, J=7.6, 1.1 Hz, 1H), 6.70 (appt, J=7.6, 1.1 Hz, 1H), 5.74 (m, 2H), 4.58 (app d, J=4.7 Hz, 2H), 4.33(m, 1H), 3.40 (m, 2H), 2.02-1.75 (m, 4H), 1.43 (s, 9H) ppm.

B. 2S-Benzofuran-3-ylmethyl-pyrrolidine-1-carboxylic acid tert-butylester (24)

To a solution of 23 (15.5 g, 36.1 mmol) in anhydrous DMF (250 mL) wasadded n-Bu₄NCl (10.0 g, 36.1 mmol), K₂CO₃ (5.0 g, 36.1 mmol), NaHCO₃(2.45 g, 36.1 mmol), and Pd(OAc)₂ (0.16 g, 0.72 mmol). The orange-brownreaction mixture was immersed in a pre-heated (100° C.) oil bath. After2 h, the reaction mixture was cooled to ambient temperature then dilutedwith diethyl ether and water. The layers were separated and the aqueousphase was extracted twice with diethyl ether. The combined organicextract was washed with water, brine, dired over anhydrous Na₂SO₄,filtered, and concentrated. The crude benzofuran was purified by flashsilica gel chromatography (4:1 hexane/EtOAc) and then by normal-phaseHPLC (30% EtOAc/hexane) to afford 4.6 g (42%) of 24 as a colorless oil.¹H NMR (CDCl₃, 300 MHz) δ 7.62 (m, 1H), 7.46 (m, 1H), 7.26 (m, 3H), 4.13(m, 1H), 3.40-3.21 (m, 2H), 3.15 (m, 1H), 2.68 (m, 1H), 1.80-1.61 (m,4H), 1.51 (s, 9H) ppm.

C. 2S-Benzofuran-3-ylmethyl-pyrrolidine (25)

Trifluoroacetic acid (2 mL) was added at 0° C. to a solution of 24 (0.5g, 1.65 mmol) in DCM (40 mL). After 2 h, an additional portion of TFA (2mL) was added. After 1 h, the reaction was quenched by the carefuladdition of aqueous NaHCO₃. The reaction mixture was diluted with waterand the crude product was extracted with diethyl ether. The organicextract was washed with aqueous NaHCO₃, brine, dried over anhydrousNa₂SO₄, filtered, and concentrated. The crude amine was purified byreverse-phase HPLC (C18; 10-100% ACN/water, 0.1% TFA) which, followingneutralization, afforded 0.24 g (72%) of 25 as a colorless oil. ¹H NMR(CDCl₃, 300 MHz) δ 7.53 (app d, J=8.7 Hz, 2H), 7.43 (d, J=8.2 Hz, 1H),7.27 (dd, J=7.0, 8.2 Hz, 1H), 7.21 (dd, J=7.0, 8.7 Hz, 1H), 6.96 (br s,1H), 3.59 (m, 1H), 3.16-2.86 (m, 4H), 1.96-1.75 (m, 3H), 1.60 (m, 1H)ppm.

D.[1S-(2S-Benzofuran-3-ylmethyl-pyrrolidine-1-carbonyl)-2-methyl-propyl]-carbamicacid tert-butyl ester (26)

A solution of N-Boc L-valine (121 mg, 0.56 mmol) in NMP (3 mL) wastreated with HATU (182 mg, 0.48 mmol) followed by N-methylmorpholine(0.1 mL, 0.9 mmol) at room temperature. After 10 min, amine 25 (80 mg,0.4 mmol) in NMP (5 mL) was added dropwise. After 16 h, the reaction wasdiluted with EtOAc, washed with saturated NaHCO₃, 1M HCl, brine, driedover Na₂SO₄, filtered and concentrated. The residual oil was purified byHPLC (20% EtOAc/hexane) to afford 111 mg (69%) of 26 as a colorless oil.¹H NMR (CDCl₃, 300 MHz) δ 7.87 (d, J=6.9 Hz, 1H), 7.48-7.32 (m, 2H),7.30-7.24 (m, 4H), 5.35 (d, J=9.3 Hz, 1H), 4.53-4.47 (m, 1H), 4.31 (dd,J=6.6. 9.6 Hz, 1H), 3.72-3.56 (m, 2H), 3.31 (dd, J=3.0, 13.2 Hz, 1H),2.49 (dd, J=10.5, 13.5 Hz, 1H), 2.05-1.74 (m, 6H), 1.44 (s, 9H), 1.03(d, J=6.9 Hz, 3H), 097 (d, J=6.6 Hz, 3H) ppm.

E.2S-Amino-1-(2S-benzofuran-3-ylmethyl-pyrrolidin-1-yl)-3-methyl-butan-1-one(27)

A solution of carbamate 26 (111 mg, 0.28 mmol) in DCM (10 mL) wastreated with TFA (1 mL) at room temperature. After 2 h, the reactionmixture was concentrated, diluted with EtOAc, washed with saturatedaqueous NaHCO₃ and brine, dried over Na₂SO₄, filtered and concentratedto afford 67 mg (80%) of 27 as a light yellow oil. The product was usedwithout further purification. ¹H NMR (CDCl₃, 300 MHz) δ 7.89 (d, J=8.1Hz, 1H), 7.47-7.44 (m, 2H), 7.32-7.27 (m, 3H), 4.54-4.49 (m, 1H),3.59-3.53 (m, 2H), 3.33 (d, J=13.5 Hz, 1H), 2.50 (dd, J=10.8, 13.5 Hz,1H), 2.03-1.72 (m, 6H), 1.04 (d, J=7.2 Hz, 3H), 0.99 (d, J=7.0 Hz, 3H)ppm.

F. {1S-[1S-(2S-Benzofuran-3-ylmethyl-pyrrolidine-1-carbonyl)-2-methyl-propylcarbamoyl]-ethyl}-carbamicacid tert-butyl ester (28)

A solution of N-Boc L-alanine (46 mg, 0.24 mmol) in NMP (3 mL) wastreated with HATU (72 mg, 0.19 mmol) followed by N-methylmorpholine (0.1mL, 0.9 mmol) at room temperature. After 10 min, amine 27 (41 mg, 0.14mmol) in NMP (5 mL) was added dropwise. After 16 h, the reaction wasdiluted with EtOAc, washed with saturated NaHCO₃, 1M HCl, and brine,dried over Na₂SO₄, filtered and concentrated. The residual oil waspurified by flash silica gel chromatography (1:1 hexane/EtOAc) to afford62 mg (93%) of 28 as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.85 (d,J=6.6 Hz, 1H), 7.48-7.44 (m, 2H), 7.33-7.25 (m, 3H), 6.93 (d, J=8.7 Hz,1H), 5.08 (d, J=7.8 Hz, 1H), 4.62 (dd, J=6.3, 8.7 Hz, 1H), 4.50-4.43 (m,1H), 4.23-4.16 (m, 1H), 3.74-3.55 (m, 3H), 3.32 (dd, J=2.4, 14.1 Hz,1H), 2.49 (dd, J=10.8, 13.5 Hz, 1H), 2.14-1.71 (m, 6H), 1.46 (s, 9H),1.37 (d, J=6.9 Hz, 3H), 1.02 (d, J=6.9 Hz, 3H), 0.97 (d, J=7.2 Hz, 3H)ppm.

G.2S-Amino-N-[1S-(2S-benzofuran-3-ylmethyl-pyrrolidine-1-carbonyl)-2-methyl-propyl]-propionamide(29)

A solution of carbamate 28 (62 mg, 0.13 mmol) in DCM (10 mL) was treatedwith TFA (1 mL) at room temperature. After 1.5 h, the reaction wasconcentrated, diluted with EtOAc, washed with saturated NaHCO₃ andbrine, dried over Na₂SO₄, filtered and concentrated. The residue waspurified by flash silica gel chromatography (5% MeOH/DCM) to afford 38mg (72%) of 29 as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.92 (d,J=9.3 Hz, 1H), 7.87-7.84 (m, 1H), 7.48-7.44 (m, 2H), 7.32-7.23 (m, 2H),4.59 (dd, J=6.9, 9.3 Hz, 1H), 4.52-4.45 (m, 1H), 3.84-3.76 (m, 1H),3.67-3.60 (m, 1H), 3.57-3.50 (m, 1H), 3.34 (dd, J=3.0, 13.0 Hz, 1H),2.50 (dd, J=10.5, 13.8 Hz, 1H), 2.16-1.91 (m, 3H), 1.82-1.69 (m, 2H),1.65 (bs, 2H), 1.38 (d, J=7.2 Hz, 3H), 1.03 (d, J=6.9 Hz, 3H), 0.99 (d,J=6.9 Hz, 3H) ppm. The free amine (29) was diluted with Et₂O and treatedwith HCl (g) to form a white solid. The solution was concentrated andthe solid was triturated with Et₂O and hexanes to provide 29.HCl.

TABLE 5 R₁₁ MS K_(D) Entry A₁ R_(1b) Y Z R₁₄₋₁₇ (M + H)⁺ Range 5-39 H MeiPr (S)—OPh H 464.2 A 5-40 H Me tBu (S)—OPh H 478.3 A 5-41 H Me iPr H H372.2 A 5-42 H Me cHex (S)—OPh H 504.2 A 5-43 Me Me cHex (S)—OPh H 518.2A 5-44 H Et iPr (S)—OPh H 478.2 A 5-45 Me Me iPr (S)—(S)—OPh H 478.2 A5-46 H H iPr H H 358.3 D 5-47 H Me cHex H H 412.3 A 5-48 Me Me cHex H H426.2 A 5-49 H Me iPr H 5-Me 386.3 A (R₁₅) 5-50 Me Me iPr H 5-Me 400.3 A(R₁₅) 5-51 Me Me iPr H 5-F 404.2 A (R₁₅) 5-52 H H iPr (S)—OPh H 450.2 C5-53 H (R)—Me iPr (S)—OPh H 464.2 C 5-54 H Me Et (S)—OPh H 450.2 A 5-55H Et Et (S)—OPh H 464.2 B 5-56 H Et cHex (S)—OPh H 518.2 B 5-57 Me MeiPr H H 386.3 A 5-58 H Et iPr H H 386.2 B 5-59 H Me Et H H 358.3 B 5-60H Me —CH(Me)OCH₂C≡CH H H 412.2 A 5-61 H Me iPr H 5-Cl 406.2 A (R₁₅) 5-62Me Me iPr H 5-Cl 420.2 A (R₁₅) 5-63 H Me iPr H 5-BnO 478.2 B (R₁₅) 5-64H gem- iPr H H 386.3 D di-Me 5-65 Ac Me iPr H H 414.4 D 5-66 H₂NCO MeiPr H H 416.2 D 5-67 HCO Me iPr H H 400.3 D 5-68 Et Me iPr H H 400.3 C5-69 iPr Me iPr H H 413.3 C 5-70 cPrCH₂ Me iPr H H 426.2 C 5-71 HC≡CCH₂Me iPr H H 409.2 D 5-72 (CH₂)₂ iPr H H 384.3 B 5-73 CH₂ iPr H H 370.2 D5-74 (CH₂)₃ iPr H H 398.4 D

Example 7

This example illustrated pyrrolidine derivatives of Table 6.

Preparation of2-Amino-N-{1-[2-(5-hydroxy-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-propionamide(32) A.(1-{1-[2-(5-Hydroxy-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propylcarbamoyl}-ethyl)-carbamicacid tert-butyl ester (31)

A mixture of 30 (0.52 g, 0.90 mmol) and 10% Pd-on-carbon (0.05 g) inEtOAc (25 mL) was placed on a Parr apparatus and pressurized to 45 PSIH₂ atmosphere. The reaction mixture was shaken for 24 h. TLC analysisrevealed only unconsumed starting material therefore the catalyst wasremoved by filtration and the clarified filtrate was concentrated todryness. The residue was redissolved in EtOAc (25 mL) and 10%Pd-on-carbon (0.208 g) was added. The reaction mixture was shaken undera H₂ atmosphere (45 PSI) for 4 h at which time a second portion ofpalladium catalyst (0.05 g) was added. Hydrogenation was continued untilall of the starting material was consumed (˜2 h). The catalyst wasremoved by filtration and the filtrate was concentrated under reducedpressure. The crude product was purified by flash silica gelchromatography (EtOAc/hexane, 1:1) to afford 0.37 g (84%) of 31 as awhite solid. ¹H NMR (CDCl₃, 300 MHz) δ7.37 (s, 1H); 7.33-7.31 (m, 1H);7.28-7.26 (m, 1H), 6.88-6.85 (m, 2H); 5.18 (br, 1H); 4.56 (t, 1H);4.45-4.24 (m, 3H); 3.82-3.74 (m, 1H); 3.66-3.58 (m, 1H); 3.22 (d, 1H);2.44-2.36 (m, 1H); 2.12-2.09 (m, 1H); 2.03-1.89 (m, 2H); 1.73-1.67 (m,2H); 1.45 (s, 9H); 1.37 (d, 3H); 1.02-0.97 (m, 6H) ppm.

B.2-Amino-N-{1-[2-(5-hydroxy-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-propionamide(32)

To a solution of 31 (0.04 g, 0.082 mmol) in DCM (5 mL) was added TFA (1mL) at ambient temperature. After 1 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was purified by flash silica gelchromatography (10% MeOH/DCM) to afford 0.011 g (37%) of 32 as a whitesolid. ¹H NMR (CDCl₃, 300 MHz) 88.54 (d, 1H); 7.37 (s, 1H); 7.31 (d,1H); 7.09 (d, 1H); 6.83 (dd, 1H); 4.64 (t, 1H); 4.56-4.51 (m, 1H);4.09-3.93 (m, 2H); 3.73-3.65 (m, 1H); 3.36 (dd, 1H); 2.35 (t, 1H);2.19-1.94 (m, 3H); 1.77-1.69 (m, 2H); 1.33 (d, 2H); 1.25-1.19 (m, 1H);1.04-1.00 (m, 5H); 0.94 (t, 1H) ppm.

TABLE 6 R_(11, 12), MS K_(D) Entry A₁ R_(1b) Y Z R₁₄₋₁₇ (M + H)⁺ Range6-75 H Me iPr H 5-OH 388.3 C (R₁₅)

Example 8

This example illustrated pyrrolidine derivatives of Table 7.

Preparation of2-Amino-N-{1-[2-(5-methoxy-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-propionamide(34) A.(1-{1-[2-(5-Methoxy-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propylcarbamoyl}-ethyl)-carbamicacid tert-butyl ester (33)

To a solution of 32 (0.06 g, 0.12 mmol) in anhydrous DCM (3 mL) wasadded MeOH (10 μL), Ph₃P (0.036 g, 0.13 mmol), and ADDP (0.037 g, 0.14mmol). The reaction mixture was maintained at ambient temperature for 16h at which point TLC analysis revealed incomplete reaction. A secondportion of MeOH (100 μL), Ph₃P (0.036 g, 0.13 mmol), and ADDP (0.037 g,0.14 mmol) was added and the reaction mixture was maintained for anadditional 5 h at ambient temperature. The reaction mixture was dilutedwith DCM and washed twice with 1 N NaOH, followed by brine, dried overanhydrous Na₂SO₄, filtered and concentrated. The crude product waspurified by flash silica gel chromatography (EtOAc/hexane, 2:1) toafford 0.057 g (91%) of 33 as a white solid. ¹H NMR (CDCl₃, 300 MHz)δ7.41 (s, 1H); 7.38-7.33 (m, 1H); 7.30-7.28 (m, 1H); 6.90 (dd, 1H); 6.77(d, 1H); 4.98 (br, 1H); 4.64-4.59 (m, 1H); 4.48-4.43 (m, 1H); 4.21-4.14(m, 1H); 3.88 (s, 3H); 3.72-3.53 (m, 2H); 3.30-3.25 (m, 1H); 2.51-2.43(m, 1H); 2.13-1.72 (m, 5H); 1.45 (s, 9H); 1.37 (d, 3H); 1.03-0.95 (m,6H) ppm.

B.2-Amino-N-{1-[2-(5-methoxy-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-propionamide(34)

To a solution of 33 (0.057 g, 0.11 mmol) in DCM (5 mL) was added TFA (1mL) at ambient temperature. After 1 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was purified by flash silica gelchromatography (10% MeOH/DCM) to afford 0.015 g (35%) of 34 as a whitesolid. ¹H NMR (CDCl₃, 300 MHz) δ7.42 (m, 1H); 7.35 (d, 1H); 7.28 (m,1H); 6.90 (dd, 1H); 4.60 (m, 1H); 4.48-4.45 (m, 1H); 3.89-3.76 (m, 4H);3.65-3.56 (m, 2H); 3.31 (dd, 1H); 2.52-2.44 (m, 1H); 2.13-1.72 (m, 6H);1.43-1.27 (m, 5H); 1.05-0.97 (m, 6H) ppm.

TABLE 7 R_(11, 12), MS K_(D) Entry A₁ R_(1b) Y Z R₁₄₋₁₇ (M + H)⁺ Range7-76 H Me iPr H 5-MeO 402.2 C (R₁₅) 7-77 H Me iPr H 5-cPrCH₂O 442.2 D(R₁₅)

Example 9

This example illustrated pyrrolidine derivatives of Table 8.

Preparation of2-Amino-N-{2-methyl-1-[2-(5-pyridin-2-yl-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-propyl}-propionamide(37) A. Trifluoro-methanesulfonic acid3-{1-[2-(2-tert-butoxycarbonylamino-propionylamino)-3-methyl-butyryl]-pyrrolidin-2-ylmethyl}-benzofuran-5-ylester (35)

To a solution of 32 (0.16 g, 0.32 mmol) and DIPEA (60 μL, 0.32 mmol) inanhydrous DCM (4 mL) was added N-phenyl-bis(trifluoromethanesulfonimide)(0.14 g, 0.38 mmol) at 0° C. After ˜10 min, the reaction mixture wasallowed to warm to ambient temperature and then maintained for 16 h. Thereaction mixture was concentrated and the residue was dissolved inEtOAc, washed with water, brine, dried over anhydrous Na₂SO₄, filteredand concentrated. The crude product was purified by flash silica gelchromatography (EtOAc/hexane, 1:1) to afford 126 mg (63%) of 35 as awhite solid. ¹H NMR (CDCl₃, 300 MHz) δ7.73 (d, 1H); 7.55-7.48 (m, 2H);7.20 (dd, 1H); 6.78-6.75 (m, 1H); 4.99 (br, 1H); 4.63-4.58 (m, 1H);4.41-4.35 (m, 1H); 4.19 (m, 1H); 3.75-3.68 (m, 1H); 3.64-3.57 (m, 1H);3.29 (dd, 1H); 2.54-2.46 (m, 1H); 1.99-1.84 (m, 3H); 1.71-1.66 (m, 2H);1.45 (s, 9H); 1.37 (d, 3H); 1.02-0.94 (m, 6H) ppm.

B.(1-{2-Methyl-1-[2-(5-pyridin-3-yl-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-propylcarbamoyl}-ethyl)-carbamicacid tert-butyl ester (36)

A mixture of 35 (0.063 g, 0.10 mmol), K₂CO₃ (70 mg, 0.50 mmol),3-pyridineboronic acid (0.015 g, 0.12 mmol), and (Ph₃P)₄Pd (5 mg, 3 mol%) in anhydrous toluene (5 mL) was heated at 100° C. in a sealed tubefor 1 h. The reaction mixture was cooled to room temperature, dilutedwith EtOAc, washed with saturated aqueous NaHCO₃, brine, dried overanhydrous Na₂SO₄, filtered and concentrated. The crude product waspurified by flash silica gel chromatography (EtOAc) to afford 40 mg(72%) of 36 as an oil. ¹H NMR (CDCl₃, 300 MHz) δ8.94 (d, 1H); 8.59 (d,1H); 8.01-7.95 (m, 1H); 7.74-7.64 (m, 1H); 7.58-7.37 (m, 4H); 6.94 (d,1H); 4.61 (t, 1H); 4.50-4.47 (m, 1H); 4.30 (t, 1H0; 4.21 (br, 1H);3.79-3.71 (m, 1H); 3.66-3.59 (m, 1H); 2.42-3.36 (m, 1H); 2.58-2.50 (m,1H); 2.13-2.09 (m, 1H); 2.01-1.71 (m, 4H); 1.45 (m, 9H); 1.37 (d, 3H);1.03-0.94 (m, 6H) ppm.

C.2-Amino-N-{2-methyl-1-[2-(5-pyridin-2-yl-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-propyl}-propionamide(37)

To a solution of 36 (0.04 g, 0.07 mmol) in DCM (5 mL) was added TFA (1mL) at ambient temperature. After 1 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was purified by flash silica gelchromatography (10% MeOH/DCM) to afford 0.024 g (76%) of 37 as a whitesolid. ¹H NMR (CDCl₃, 300 MHz) δ8.0 (s, 1H); 7.95 (d, 1H); 7.71-7.64 (m,2H); 7.56-7.46 (m, 4H); 4.61-4.48 (m, 2H); 4.29 (t, 1H); 3.83-3.77 (m,1H); 3.67-3.54 (m, 2H); 3.41 (dd, 1H); 2.58-2.49 (m, 1H); 2.13-1.70 (m,5H); 1.40-1.25 (m, 5H); 1.04-0.84 (m 6H) ppm.

TABLE 8 R_(11, 12), MS K_(D) Entry A₁ R_(1b) Y Z R₁₄₋₁₇ (M + H)⁺ Range8-78 H Me iPr H 5-(4-Pyr) 449.2 B (R₁₅) 8-79 H Me iPr H 5-(3-Pyr) 449.2C (R₁₅)

Example 10

This example illustrated pyrrolidine derivatives of Table 9.

The Preparation of2S-Amino-N-{2-methyl-1S-[2S-(2-pyridin-4-yl-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-propyl}-propionamide(44) A. 2S-(2-Bromo-benzofuran-3-ylmethyl)-pyrrolidine-1-carboxylic acidtert-butyl ester (38)

A solution of 22 (1.4 g, 4.7 mmol) in CHCl₃ (20 mL) was cooled to 0° C.and treated with KOAc (2.5 g, 26 mmol). A solution of bromine (0.3 mL,5.8 mmol) in CHCl₃ (10 mL) was added dropwise over 30 min. Followingaddition, the solution maintained a yellow orange color. After TLCindicated consumption of starting material, the reaction mixture wasdiluted with H₂O, washed with saturated Na₂S₂O₈, dried over Na₂SO₄,filtered, and concentrated. Purification of the residue by flash silicagel chromatography (3:1 hexane/EtOAc) afforded 1.1 g (61%) of 38 as ayellow oil. ¹H NMR (CDCl₃, 300 MHz) δ 7.57-7.52 (m, 1H), 7.43-7.41 (m,1H), 7.26-7.20 (m, 2H), 4.20 (m, 1H), 3.43-3.30 (m, 2H), 3.17-3.01 (m,1H), 2.73-2.58 (m, 1H), 1.89-1.69 (m, 4H), 1.48 (s, 9H) ppm.

B. 2S-(2-Bromo-benzofuran-3-ylmethyl)-pyrrolidine (39)

A solution of 38 (1.1 g, 2.9 mmol) in DCM (20 mL) was treated with TFA(2 mL) at room temperature. After 1.5 h, the reaction mixture wasconcentrated, diluted with EtOAc, washed with saturated NaHCO₃, brine,dried over Na₂SO₄, filtered and concentrated to afford 0.8 g of 39 as anorange oil which was used without further purification. ¹H NMR (CDCl₃,300 MHz) δ 8.84 (br s, 1H), 7.42-7.39 (m, 2H), 7.28-7.21 (m, 2H),3.70-3.66 (m, 1H), 3.48-3.39 (m, 1H), 3.31-3.21 (m, 2H) 2.95 (dd, J=9.9Hz, 13.5 Hz, 1H), 2.06-1.99 (m, 1H), 1.91-1.79 (m, 2H), 1.74-1.66 (m,1H) ppm.

C.{1S-[2S-(2-Bromo-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-carbamicacid tert-butyl ester (40)

A solution of N-Boc L-valine (656 mg, 3.0 mmol) in NMP (6 mL) wastreated with HATU (1.2 g, 3.1 mmol) followed by N-methylmorpholine (0.4mL, 3.6 mmol) at room temperature. After 10 min, crude 39 (0.8 g, 2.9mmol) in NMP (6 mL) was added dropwise. After 16 h, the reaction mixturewas diluted with EtOAc, washed with saturated NaHCO₃, brine, dried overNa₂SO₄, filtered and concentrated. The residue was purified by flashsilica gel chromatography (3:1 hexane/EtOAc) to afford 853 mg (61%) of40 as a yellow foam. ¹H NMR (CDCl₃, 300 MHz) δ 7.89-7.86 (m, 1H),7.42-7.38 (m, 1H), 7.29-7.25 (m, 3H), 5.35 (d, J=9.3 Hz, 1H), 4.55-4.51(m, 1H), 4.29 (dd, J=6.0, 9.6 Hz, 1H), 3.71-3.66 (m, 1H), 3.27 (dd,J=3.3, 13.5 Hz, 1H), 2.45 (dd, J=10.8, 13.5 Hz, 1H), 2.15-1.92 (m, 3H),1.79-1.74 (m, 2H), 1.42 (s, 9H), 1.03 (d, J=6.9 Hz, 3H), 0.96 (d, J=7.2Hz, 3H) ppm.

D.2S-Amino-1-[2S-(2-bromo-benzofuran-3-ylmethyl)-pyrrolidin-1-yl]-3-methyl-butan-1-one(41)

A solution of carbamate 40 (853 mg, 1.8 mmol) in DCM (20 mL) was treatedwith TFA (2 mL) at room temperature. After 1.5 h, the reaction mixturewas concentrated, diluted with EtOAc, washed with saturated NaHCO₃,brine, dried over Na₂SO₄, filtered and concentrated to afford 0.66 g of41 as a yellow foam which was used without further purification. ¹H NMR(CDCl₃, 300 MHz) δ 7.92-7.89 (m, 1H), 7.44-7.39 (m, 1H), 7.30-7.25 (m,3H), 4.55 (m, 1H), 3.66-3.51 (m, 2H), 3.29 (dd, J=2.4, 13.5 Hz, 1H),2.45 (dd, J=11.1, 12.9 Hz, 1H), 2.15-1.97 (m, 2H), 1.78-1.71 (m, 2H),1.06 (d, J=6.3 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H) ppm.

E.(1S-{1S-[2S-(2-Bromo-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propylcarbamoyl}-ethyl)-carbamicacid tert-butyl ester (42)

A solution of N-Boc-L-alanine (394 mg, 2.1 mmol) in NMP (6 mL) wastreated with HATU (763 mg, 2.0 mmol) followed by N-methylmorpholine (0.3mL, 2.7 mmol) at room temperature. After 10 min, crude 41 (0.66 mg, 1.7mmol) in NMP (6 mL) was added dropwise. After 16 h, the reaction mixturewas diluted with EtOAc, washed with saturated NaHCO₃, 1M HCl, brine,dried over Na₂SO₄, filtered and concentrated. The residual oil waspurified by flash silica gel chromatography (2:1 hexane/EtOAc-to-1:1hexane/EtOAc) to afford 0.9 g (96%) of 42 as an off-white foam. ¹H NMR(CDCl₃, 300 MHz) δ 7.86-7.84 (m, 1H), 7.43-7.40 (m, 1H), 7.29-7.25 (m,2H), 7.01 (d, J=8.7 Hz, 1H), 5.10 (d, J=6.6 Hz, 1H), 4.62 (dd, J=6.3,8.7 Hz, 1H), 4.53-4.48 (m, 1H), 4.23-4.19 (m, 1H), 3.75-3.66 (m, 2H),3.27 (dd, J=3.3, 13.5 Hz, 1H), 2.45 (dd, J=10.5, 13.5 Hz, 1H), 2.16-2.08(m, 2H), 2.02-1.97 (m, 2H), 1.79-1.73 (m, 2H), 1.45 (s, 9H), 1.36 (d,J=6.9 Hz, 3H), 1.02 (d, J=6.3 Hz, 3H), 0.97 (d, J=7.2 Hz, 3H) ppm.

F.(1S-{2-Methyl-1S-[2S-(2-pyridin-4-yl-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-propylcarbamoyl}-ethyl)-carbamicacid tert-butyl ester (43)

A solution of 42 (36 mg, 0.07 mmol) in toluene (4 mL) was treated withK₂CO₃ (38 mg, 0.28 mmol) and Pd(PPh₃)₄ (2 mg) followed by 4-pyridineboronic acid (12 mg, 0.1 mmol) in EtOH (2 mL). The reaction was heatedto reflux overnight. The solution was cooled to room temperature,diluted with H₂O, extracted with EtOAc, brine, dried over Na₂SO₄,filtered and concentrated to afford 44 mg of 43 as a light yellow oilwhich was used without further purification. ¹H NMR (CDCl₃, 300 MHz) δ8.74 (d, J=5.1 Hz, 1H), 7.99 (d, J=5.7 Hz, 1H), 7.86-7.81 (m, 1H),7.71-7.64 (m, 1H), 7.56-7.46 (m, 3H), 7.41-7.27 (m, 2H), 6.87 (d, J=8.7Hz, 1H), 5.01 (m, 1H), 4.62 (app t, J=8.7 Hz, 1H), 4.56-4.46 (m, 1H),4.20 (m, 1H), 3.72-3.67 (m, 2H), 2.84 (dd, J=11.7, 13.5 Hz, 1H),2.16-1.93 (m, 2H), 1.78-1.62 (m, 2H), 1.46 (s, 9H), 1.37 (d, J=7.2 Hz,3H), 1.04 (d, J 6.6 Hz, 3H), 0.98 (d, J=6.3 Hz, 3H) ppm.

G.2S-Amino-N-{2-methyl-1S-[2-(2S-pyridin-4-yl-benzofuran-3-ylmethyl)-pyrrolidine-1-carbonyl]-propyl}-propionamide(44)

A solution of 43 (44 mg, 0.08 mmol) in DCM (10 mL) was treated with TFA(1 mL) at room temperature. After 1.5 h, the reaction mixture wasconcentrated, diluted with EtOAc, washed with saturated NaHCO₃, brine,dried over Na₂SO₄, filtered and concentrated. The residue was purifiedby reverse-phase HPLC (C18; 10-100% ACN/H₂O with 0.1% AcOH buffer). Thefractions containing pure product were lyophilized to afford 21 mg of 44as a white solid. ¹H NMR (CDCl3, 300 MHz) δ 8.75 (bs, 1H), 8.00 (m, 1H),7.90-7.82 (m, 1H), 7.71-7.64 (m, 1H), 7.55-7.47 (m, 3H), 7.38-7.27 (m,2H), 4.61-4.52 (m, 1H), 3.83-3.62 (m, 2H), 3.07-3.00 (m, 1H), 2.84 (dd,J=11.1, 13.5 Hz, 1H), 2.16-1.94 (m, 2H), 1.74-1.61 (m, 2H), 1.41 (d,J=6.9 Hz, 3H), 1.05 (d, J=6.6 Hz, 3H), 0.99 (d, J=6.9 Hz, 3H) ppm.

TABLE 9 MS K_(D) Entry A₁ R_(1b) Y Z X₂ R₁₁ (M + H)⁺ Range 9-80 H Me iPrH O Br 450.4 B 9-81 H Me iPr H O 4-Pyr 449.2 A 9-82 H Me iPr H O 3-Pyr449.2 B 9-83 H Me iPr H O 5-[(2- 479.2 B MeO)Pyr] 9-84 H Me iPr H O2-Pyr 449.3 A 9-85 H Me iPr H O 2-thiazoyl 455.2 B 9-86 H Me iPr H O2-pyrazine 450.1 A 9-87 H Me iPr H O Ph 448.3 B 9-88 H Me iPr H N Ph,(R₁₂ is H) 447.2 C

Example 11

This example illustrated pyrrolidine derivatives of Table 10.

The Preparation of3-(3-{1-[2-(2-Amino-propionylamino)-3-methyl-butyryl]-pyrrolidin-2-ylmethyl}-benzofuran-2-yl)-propionicacid ethyl ester (52) A.2-(2-Bromo-benzofuran-3-ylmethyl)-pyrrolidine-1-carboxylic acid benzylester (46)

To a cooled (0° C.) solution of 45 (4.1 g, 12.2 mmol) in CHCl₃ (70 mL)was added KOAc (3.6 g, 36.7 mmol) followed by the dropwise addition ofBr₂ (2.3 g, 14.6 mmol) in CHCl₃ (30 mL) over ˜20 min. After 30 min, thereaction mixture was diluted with brine and saturated with Na₂S₂O₈. Theproduct was extracted with DCM and the combined DCM extracts were washedwith brine, dried over anhydrous Na₂SO₄, filtered and concentrated toafford 5 g (quant.) of 46 as an orange oil which was used withoutfurther purification. ¹H NMR (CDCl₃, 300 MHz) δ 7.75-7.72 & 6.98(rotamers, m and app t, J=7.5 Hz, 1H), 7.44-7.10 (m, 8H), 5.24-5.17 (m,2H), 4.28 & 4.17 (rotamer, 2 m, 1H), 3.54-3.38 (m, 2H), 3.21 & 3.00(rotamer, 2 dd, J=2.4, 13.5 Hz, 1H), 2.72-2.55 (m, 1H), 1.97-1.75 (m,4H) ppm.

B.2-[2-(2-Ethoxycarbonyl-vinyl)-benzofuran-3-ylmethyl]-pyrrolidine-1-carboxylicacid benzyl ester (47)

A solution containing crude 46 (5.0 g, 12.1 mmol), ethyl acrylate (2.7g, 27.7 mmol), tetrabutylammonium chloride (3.5 g, 12.6 mmol), NaHCO₃(2.0 g, 23.8 mmol), and Pd(OAc)₂ (0.107 g, 0.46 mmol) in DMF (40 mL) washeated at 100° C. for 16 h. After cooling to ambient temperature, thereaction mixture was diluted with diethyl ether and washed with water,brine, dried over anhydrous Na₂SO₄, filtered and concentrated. The crudeproduct was purified by flash silica gel chromatography (EtOAc/hexane,1:3) to afford 3.7 g (70%) of 47 as an orange-colored oil. ¹H NMR(CDCl₃, 300 MHz) δ7.81 & 6.99 (rotamer, app d, J=7.5 Hz, app t, J=7.5Hz, 111), 7.61 (dd, J=15.9, 18.3 Hz, 1H), 7.47-7.20 (m, 8H), 6.58 (dd,J=5.7, 15.6 Hz, 1H), 5.27-5.16 (m, 2H), 4.24 (q, J=7.2 Hz, 2H),4.17-4.08 (m, 1H), 3.51-3.35 (m, 2H), 3.38 & 3.17 (rotamer, 2 dd, J=3.6,14.1 Hz, 1H), 2.89-2.69 (m, 1H), 1.94-1.64 (m, 4H), 1.36 (t, J=7.2 Hz,2H) ppm.

C. 3-(3-Pyrrolidin-2-ylmethyl-benzofuran-2-yl)-propionic acid ethylester (48)

A mixture containing 47 (1.0 g, 2.3 mmol) and 10% Pd-on-carbon (˜1 g) inanhydrous MeOH (20 mL) was placed under a hydrogen atmosphere (45 PSI)and shaken using a Parr apparatus for 1 h. The catalyst was removed byfiltration and the solids were washed with EtOAc and MeOH. The clarifiedfiltrate was concentrated to afford 634 mg (91%) of 48 as an orange oilwhich was used without further purification. ¹H NMR (CDCl₃, 300 MHz)δ7.51-7.49 (m, 1H), 7.38-7.35 (m, 1H), 7.21-7.17 (m, 2H), 4.16-4.09 (m,2H), 3.68-3.59 (m, 2H), 3.12-3.07 (m, 2H), 2.88-2.73 (m, 4H), 1.84-1.69(m, 4H), 1.43-1.39 (m, 1H), 1.26-1.20 (m, 3H) ppm.

D.3-{3-[1-(2-tert-Butoxycarbonylamino-3-methyl-butyryl)-pyrrolidin-2-ylmethyl]-benzofuran-2-yl}-propionicacid ethyl ester (49)

A solution containing Boc-L-valine (352 mg, 1.6 mmol) in NMP (3 mL) wastreated with HATU (543 mg, 1.4 mmol) followed by NMM (0.2 mL, 1.8 mmol)at ambient temperature. After 10 min, amine 48 (0.4 g, 1.3 mmol) in NMP(5 mL) was added in a dropwise fashion. After 16 h, the reaction mixturewas diluted with diethyl ether and washed successively with NaHCO₃, 1 MHCl, water, and brine. The organic extract was dried over anhydrousNa₂SO₄, filtered and concentrated to afford 630 mg (96%) of 49 which wasused without further purification. ¹H NMR (CDCl₃, 300 MHz) δ7.82-7.79(m, 1H), 7.38-7.35 (m, 1H), 7.28-7.21 (m, 2H), 5.37 (d, J=9.3 Hz, 1H),4.50-4.44 (m, 1H), 4.31 (dd, J=6.0, 9.0 Hz, 1H), 4.14 (q, J=6.9 Hz, 2H),3.72-3.60 (m, 3H), 3.38 (t, J=7.2 Hz, 1H), 3.29 (dd, J=3.0, 13.5 Hz,1H), 3.12 (app t, J=7.5 Hz, 2H), 2.80-2.74 (m, 4H), 2.46-2.35 (m, 2H),2.11-1.92 (m, 2H), 1.81-1.73 (m, 1H), 1.69-1.65 (m, 1H), 1.44 (s, 9H),1.25 (t, J=6.0 Hz, 3H), 1.03 (d, J=6.3 Hz, 3H), 0.96 (d, J=6.6 Hz, 3H)ppm. Mass spectrum: m/z 501 [M+H]⁺; and, 523 [M+Na]⁺.

E.3-{3-[1-(2-Amino-3-methyl-butyryl)-pyrrolidin-2-ylmethyl]-benzofuran-2-yl}-propionicacid ethyl ester (50)

To a solution of 49 (302 mg, 0.6 mmol) in DCM (10 mL) was added TFA (2mL) at ambient temperature. After 2 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated to afford 235 mg (97%) of 50 as an orange oil which wasused without further purification. ¹H NMR (CDCl₃, 300 MHz) δ7.86-7.83(m, 1H), 7.43-7.34 (m, 1H), 7.27-7.19 (m, 3H), 4.49 (m, 1H), 4.14 (q,J=6.9 Hz, 2H), 3.60-3.53 (m, 2H), 3.32 (d, J=12.9 Hz, 1H), 3.12 (app t,J=7.2 Hz, 2H), 2.81-2.74 (m, 2H), 2.42 (app t, J=12.3 Hz, 1H), 2.10-1.91(m, 2H), 1.74-1.66 (m, 2H), 1.24 (t, J=7.2 Hz, 3H), 1.06-0.99 (m, 6H)ppm. Mass spectrum: m/z 401 [M+H]⁺.

F.3-(3-{1-[2-(2-tert-Butoxycarbonylamino-propionylamino)-3-methyl-butyryl]-pyrrolidin-2-ylmethyl}-benzofuran-2-yl)-propionicacid ethyl ester (51)

A solution containing Boc-L-alanine (69 mg, 0.37 mmol) in NMP (3 mL) wastreated with HATU (131 mg, 0.34 mmol) followed by NMM (0.1 mL, 0.9 mmol)at ambient temperature. After 10 min, amine 50 (111 mg, 0.28 mmol) inNMP (5 mL) was added in a dropwise fashion. After 5 h, the reactionmixture was diluted with diethyl ether and washed successively withNaHCO₃, 1 M HCl, water, and brine. The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated to afford 160 mg (quant.) of51 which was used without further purification. ¹H NMR (CDCl₃, 300 MHz)δ7.81-7.78 (m, 1H), 7.38-7.36 (m, 1H), 7.28-7.21 (m, 3H), 6.86 (d, J=8.7Hz, 1H), 5.04 (d, J=6.9 Hz, 1H), 4.61 (dd, J=6.3, 8.7 Hz, 1H), 4.48-4.42(m, 1H), 4.22 (m, 1H), 4.13 (q, J=6.9 Hz, 2H), 3.73-3.62 (m, 2H),3.41-3.36 (m, 2H), 3.28 (dd, J=3.0, 13.5 Hz, 1H), 3.11 (app t, J=6.9 Hz,2H), 2.85-2.73 (m, 4H), 2.38 (app t, J=8.1 Hz, 2H), 2.13-1.98 (m, 2H),1.80-1.64 (m, 1H), 1.45 (s, 9H), 1.37 (d, J=6.9 Hz, 3H), 1.26 (t, J=7.5Hz, 3H), 1.03-0.95 (m, 6H) ppm. Mass spectrum: m/z 572 [M+H]⁺; and, 594[M+Na]⁺.

G.3-(3-{1-[2-(2-Amino-propionylamino)-3-methyl-butyryl]-pyrrolidin-2-ylmethyl}-benzofuran-2-yl)-propionicacid ethyl ester (52)

To a solution of 51 (160 mg, 0.28 mmol) in DCM (10 mL) was added TFA (2mL) at ambient temperature. After 2.5 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. A portion (˜50%) of the crude material was purified byreverse-phase HPLC (C18, 10-100% ACN/water containing 0.1% HOAc) toafford 56 mg of 52 as a white solid. ¹H NMR (DMSO, 300 MHz) 58.03 (d,J=8.7 Hz, 1H), 7.77 (m, 1H), 7.43-7.41 (m, 1H), 7.21 (m, 2H), 4.37 (appt, J=8.1 Hz, 1H), 4.19 (m, 1H), 4.01 (q, J=7.2 Hz, 2H), 3.65 (m, 1H),3.56 (app d, J=6.6 Hz, 1H), 3.28 (m, 1H), 3.06-3.01 (m, 3H), 2.68 (appt, J=7.2 Hz, 2H), 2.07-1.96 (m, 1H), 1.63 (m, 1H), 1.54 (m, 1H),1.13-1.09 (m, 6H), 0.90 (d, J=6.3 Hz, 3H), 0.85 (d, J=6.3 Hz, 3H) ppm.Mass spectrum: m/z 472 [M+H]⁺; and, 494 [M+Na]⁺.

TABLE 10 MS K_(D) Entry A₁ R_(1b) Y Z X₂ R₁₁ (M + H)⁺ Range 10-89 H MeiPr H O —CH₂CH₂CO₂Et 472.3 C 10-90 H Me iPr H O —CH₂CH₂CO₂H 444.2 B

Example 12

This example illustrated pyrrolidine derivatives of Table 11 and Table12.

The Preparation of Ornithine-Derived Lactam (59) A.3-{3-[1-(5-Benzyloxycarbonylamino-2-tert-butoxycarbonylamino-pentanoyl)-pyrrolidin-2-ylmethyl]-benzofuran-2-yl}-propionicacid ethyl ester (53)

A solution containing Boc-L-ornithine (2.3 g, 6.3 mmol) in NMP (5 mL)was treated with HATU (2.4 g, 6.3 mmol) followed by NMM (1.5 mL, 13.7mmol) at ambient temperature. After 10 min, amine 48 (1.6 g, 5.3 mmol)in NMP (10 mL) was added in a dropwise fashion. After 16 h, the reactionmixture was diluted with diethyl ether and washed successively withNaHCO₃, 1 M HCl, water, and brine. The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated to afford 3.0 g (87%) of 53as an orange-colored oil which was used without further purification. ¹HNMR (CDCl₃, 300 MHz) δ7.78-7.75 (m, 1H), 7.40-7.20 (m, 8H), 5.48 (d,J=8.4 Hz, 1H), 5.10 (s, 2H), 4.48-4.42 (m, 1H), 4.17-4.09 (m, 2H),3.64-3.56 (m, 1H), 3.27-3.19 (m, 1H), 3.12 (app t, J=7.8 Hz, 1H),2.81-2.74 (m, 2H), 2.52-2.35 (m, 1H), 2.09-1.95 (m, 2H), 1.83-1.63 (m,4H), 1.45 (s, 9H), 1.29-1.19 (m, 6H) ppm.

B.3-{3-[1-(5-Benzyloxycarbonylamino-2-tert-butoxycarbonylamino-pentanoyl)-pyrrolidin-2-ylmethyl]-benzofuran-2-yl}-propionicacid (54)

A solution containing the crude 53 (3.0 g, 4.6 mmol) in THF (20 mL) wastreated with 3 M NaOH (8 mL) at ambient temperature. The reactionmixture was then warmed to gentle reflux and maintained for 2.5 h. Thereaction mixture was concentrated in vacuo and the residue was dissolvedin EtOAc. The organic solution was washed with 1 N HCl then dried overanhydrous Na₂SO₄, filtered and concentrated to afford ˜3 g (quant.) of54 as an off-white-colored foam which was used directly without furtherpurification. ¹H NMR (CDCl₃, 300 MHz) δ7.69-7.67 (m, 1H), 7.39-7.20 (m,8H), 5.70 (d, J=8.4 Hz, 1H), 5.13-5.09 (m, 2H), 4.50-4.45 (m, 1H),3.67-3.54 (m, 1H), 3.22-3.18 (m, 2H), 3.13 (t, J=6.9 Hz, 1H), 2.85-2.78(m, 2H), 2.09-1.97 (m, 3H), 1.76-1.62 (m, 4H), 1.43 (s, 9H) ppm.

C.3-{3-[1-(5-Amino-2-tert-butoxycarbonylamino-pentanoyl)-pyrrolidin-2-ylmethyl]-benzofuran-2-yl}-propionicacid (55)

A solution of the crude 54 (˜3 g, 4.8 mmol) in MeOH (30 mL) was chargedwith 10% Pd-on-carbon (˜1 g) and placed under an atmosphere of hydrogengas (45 PSI). The reaction mixture was shaken using a Parr apparatus for2 h. After removal of the catalyst by filtration over celite, theclarified filtrate was concentrated in vacuo. The crude residue wastaken up in DCM and dried using anhydrous Na₂SO₄, filtered andconcentrated to afford 2.3 g (quant.) of 55 as a foamy residue which wasused without further purification. ¹H NMR (CDCl₃, 300 MHz) δ7.51 (m,1H), 7.34-7.32 (m, 1H), 7.18-7.15 (m, 2H), 6.19 (m, 1H), 4.36 (m, 2H),3.62-3.48 (m, 2H), 3.10-2.99 (m, 2H), 2.85-1.81 (m, 2H), 2.66-2.60 (m,2H), 1.99-1.71 (m, 6H), 1.41 (s, 911) ppm. Mass spectrum, m/z 488[M+H]⁺.

D. Boc-Protected Des-Alanine Ornithine-Derived Lactam (56)

A solution containing 55 (2.3 g, 4.7 mmol) in 1:1 NMP/DCM (30 mL) wastreated with HATU (2.1 g, 5.5 mmol) and NMM (0.6 mL, 5.5 mmol) atambient temperature. After 18 h, the reaction mixture was diluted withdiethyl ether and washed successively with aqueous Na₂SO₄, diluteaqueous HCl, water, brine, then dried over anhydrous Na₂SO₄, filteredand concentrated to afford 1.5 g (68%) of 56 as an off-white-coloredfoam which was used without further purification. ¹H NMR (CDCl₃, 300MHz) δ7.70-7.66 (m, 1H), 7.33 (m, 1H), 7.19-7.18 (m, 2H), 5.70 (m, 1H),4.42 (m, 1H), 4.10 (m, 1H), 3.55 (m, 3H), 3.24-3.09 (m, 4H), 2.61-2.46(m, 2H), 1.63 (m, 7H), 1.44 (s, 9H) ppm. Mass spectrum, m/z 470 [M+H]⁺,492 [M+Na]⁺.

E. Des-Alanine Ornithine-Derived Lactam (57)

To a solution of crude 56 (1.5 g, 3.1 mmol) in DCM (20 mL) was added TFA(4 mL) at ambient temperature. After 2 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, and brine. The combined aqueous washes were backextracted with 5% MeOH/DCM. The combined organic extracts were driedover anhydrous Na₂SO₄, filtered and concentrated to afford 0.8 g (68%)of 57 as an off-white-colored foam which was used directly in the nextreaction. ¹H NMR (CDCl₃, 300 MHz) δ7.41-7.38 (m, 1H), 7.27-7.20 (m, 3H),5.99 (d, J=9.3 Hz, 1H), 4.39-4.33 (m, 1H), 3.96-3.84 (m, 1H), 3.70-3.65(m, 2H), 3.55-3.38 (m, 2H), 3.20-3.05 (m, 2H), 2.90-2.83 (m, 3H),2.77-2.71 (m, 2H), 2.66-2.62 (m, 2H), 2.19-1.98 (m, 2H), 1.78-1.66 (m,2H), 1.38-1.08 (m, 2H) ppm.

F. Boc-Protected Ornithine-Derived Lactam (58)

A solution containing Boc-L-alanine (526 mg, 2.8 mmol) in NMP (5 mL) wastreated with HATU (1.1 g, 2.9 mmol) followed by NMM (0.3 mL, 2.9 mmol)at ambient temperature. After 10 min, crude amine 57 (0.8 mg, 2.2 mmol)in NMP (10 mL) was added in a dropwise fashion. After 2 d, the reactionmixture was diluted with diethyl ether and washed successively withNaHCO₃, 1 M HCl, water, and brine. The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated to afford ˜1 g (84%) of 58as a yellow-colored oil which was used without further purification.Mass spectrum: m/z 541 [M+H]⁺, and 563 [M+Na]⁺.

G. Ornithine-Derived Lactam (59)

To a solution of crude 58 (˜1 g, 1.9 mmol) in DCM (10 mL) was added TFA(4 mL) at ambient temperature. After 1 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, and brine. The combined aqueous washes were backextracted with 5% MeOH/DCM and the combined organic extracts were driedover anhydrous Na₂SO₄, filtered and concentrated. A portion of the crudeproduct was purified by reverse-phase HPLC (C18, 10-100% ACN/watercontaining 0.1% HOAc) to afford 28 mg of 59 as a white solid. ¹H NMR(DMSO, 300 MHz) 88.14 (m, 1H), 7.88 (m, 1H), 7.68-7.66 (m, 1H), 7.35 (m,1H), 7.12 (m, 2H), 4.43 (m, 1H), 4.12 (m, 1H), 3.48 (m, 2H), 2.91 (m,4H), 1.48-1.36 (m, 4H), 1.10 (d, J=6.3 Hz, 3H) ppm. Mass spectrum: m/z441 [M+H]⁺.

TABLE 11 R_(11, 12), MS K_(D) Entry A₁ R_(1b) Y Z X₂ R₁₄₋₁₇ (M + H)⁺Range 11-91 H Me Y linked H O R₁₁ 441.3 B with linked R₁₁ with Y

TABLE 12 R_(11, 12), MS K_(D) Entry A₁ R1 Y Z X₂ R₁₄₋₁₇ (M + H)⁺ Range12-92 H Me Y linked H O R₁₁ 455.2 C with linked R₁₁ with Y

Example 13

This example illustrated pyrrolidine derivatives of Table 13.

The Preparation of Tyrosine-Derived Cyclic Ether (66) A.2-[2-(3-Hydroxy-propenyl)-benzofuran-3-ylmethyl]-pyrrolidine-1-carboxylicacid benzyl ester (60)

At −78° C., BF₃.etherate (0.6 mL, 4.8 mmol) was added to a solutioncontaining 47 (1.7 g, 3.9 mmol) in anhydrous DCM (40 mL). After 10 min,DIBAL (1 M/DCM, 10 mL, 10 mmol) was added dropwise from an additionfunnel. After 5 min following the complete addition of the DIBALsolution, EtOAc (10 mL) was added to quench the excess reagent. Diluteaqueous HCl was slowly added and the reaction mixture was allowed toslowly warm to ambient temperature. The product was extracted with DCMand EtOAc and the combined organic extracts were washed with diluteaqueous HCl, water, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was purified by flash silica gelchromatography (EtOAc/hexane, 1:2) to afford 1.1 g (72%) of 60 as ayellow-colored oil. ¹H NMR (CDCl₃, 300 MHz) δ7.65 & 7.03 (rotamer, d andapp t, J=6.9, 7.8 Hz, 1H), 7.38 (m, 5H), 7.26-7.19 (m, 2H), 6.70-6.52(m, 2H), 5.26-5.14 (m, 2H), 4.38 (m, 1H), 4.28 (m, 1H), 4.14 (m, 1H),3.43 (m, 2H), 3.27 & 3.02 (rotamer, 2 m, 1H), 2.76-2.68 (m, 1H), 2.45(m, 1H), 1.77-1.68 (m, 5H) ppm.

B. 3-(3-Pyrrolidin-2-ylmethyl-benzofuran-2-yl)-propan-1-ol (61)

A mixture of 60 (1.1 g, 2.8 mmol) and 10% Pd-on-carbon (1 g) in MeOH (40mL) was placed under an atmosphere of hydrogen gas (45 PSI) and shakenfor 2 h using a Parr apparatus. The catalyst was removed by filtrationthrough a bed of celite and the clarified filtrate was concentrated invacuo to afford 725 mg (quant.) of 61 as a yellow oil which was usedwithout further purification. ¹H NMR (CDCl₃, 300 MHz) δ7.47-7.36 (m,2H), 7.21-7.17 (m, 2H), 4.59 (br s, 2H), 3.57-3.50 (m, 2H), 3.40-3.31(m, 1H), 3.08-2.94 (m, 1H), 2.89-2.69 (m, 2H), 2.05-1.95 (m, 2H),1.93-1.49 (m, 2H) ppm.

C.(1-(4-Hydroxy-benzyl)-2-{2-[2-(3-hydroxy-propyl)-benzofuran-3-ylmethyl]-pyrrolidin-1-yl}-2-oxo-ethyl)-carbamicacid tert-butyl ester (62)

A solution containing Boc-L-tyrosine (577 mg, 2.1 mmol) and HATU (716mg, 1.8 mmol) in anhydrous NMP (6 mL) was treated with NMM (0.3 mL, 2.7mmol) at ambient temperature. After 10 min, a solution containing 61(429 mg, 1.7 mmol) in NMP (5 mL) was added. After 2 d, the reactionmixture was diluted with water and the product was extracted withdiethyl ether. The combined ether extracts were washed with water anddilute aqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was purified by flash silica gelchromatography (10% MeOH/DCM) to afford 821 mg (92%) of 62 as a paleyellow foam. ¹H NMR (CDCl₃, 300 MHz) δ8.54 (br 2, 1H), 7.66-7.63 (m,1H), 7.36-7.32 (m, 1H), 7.21-7.06 (m, 5H), 6.83 (d, J=8.4 Hz, 2H), 5.58(d, J=9.0 Hz, 1H), 4.64-4.56 (m, 1H), 4.34-4.31 (m, 1H), 3.92-3.88 (m,1H), 3.70 (app t, J=5.7 Hz, 2H), 3.51-3.26 (m, 1H), 3.18-2.79 (m, 4H),2.01-1.97 (m, 2H), 1.69-1.47 (m, 1H), 1.42 (s, 9H) ppm.

D. Boc-Protected Des-Alanine Tyrosine-Derived Cyclic Ether (63)

To a solution containing 62 (821 mg, 1.6 mmol) in DCM (40 mL) was addedPh₃P (431 mg, 1.6 mmol) and ADDP (491 mg, 1.9 mmol) at ambienttemperature. After 16 h, the reaction mixture was concentrated in vacuothen redissolved in diethyl ether. The insoluble white solid was removedby filtration and the clarified filtrate was concentrated. The crudeproduct was purified by flash silica gel chromatography (EtOAc/hexane,1:2 to 1:1) to afford 160 mg (19%) of 63 as a light yellow-colored foam.¹H NMR (CDCl₃, 300 MHz) δ7.47 (d, J=7.5 Hz, 1H), 7.34 (d, J=7.8 Hz, 1H),7.28-7.24 (m, 2H), 7.22-7.11 (m, 2H), 7.00 (dd, J=2.7, 8.1 Hz, 1H), 6.69(dd, J=3.0, 8.7 Hz, 1H), 5.28 (d, J=9.6 Hz, 1H), 4.88-4.79 (m, 1H),4.51-4.41 (m, 1H), 4.23-4.17 (m, 1H), 3.97-3.89 (m, 1H), 3.84-3.76 (m,1H), 3.46-3.29 (m, 2H), 2.75-2.62 (m, 4H), 2.17-2.09 (m, 1H), 1.99-1.90(m, 1H), 1.72-1.54 (m, 2H), 1.45 (s, 9H) ppm.

E. Des-Alanine Tyrosine-Derived Cyclic Ether (64)

To a solution of 63 (160 mg, 0.32 mmol) in DCM (10 mL) was added TFA (2mL) at ambient temperature. After 1.5 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated to afford 130 mg (quant.) of 64 as an off-white-coloredsolid which was used directly in the next reaction. ¹H NMR (CDCl₃, 300MHz) δ7.46 (d, J=7.8 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 7.21-7.19 (m, 2H),7.16-7.10 (m, 2H), 6.97 (dd, J=2.4, 8.1 Hz, 1H), 6.70-6.68 (m, 1H), 4.47(m, 1H), 4.21-4.13 (m, 2H), 3.93 (t, J=9.3 Hz, 1H), 3.51-3.36 (m, 4H),2.73-2.68 (m, 4H), 2.15-2.07 (m, 1H), 1.98-1.90 (m, 1H), 1.70-1.46 (m,3H), 1.19-1.13 (m, 1H), 0.64-0.55 (m, 1H) ppm.

F. Boc-Protected Tyrosine-Derived Cyclic Ether (65)

A solution containing Boc-L-alanine (63 mg, 0.33 mmol) in NMP (3 mL) wastreated with HATU (125 mg, 0.33 mmol) followed by NMM (0.1 mL, 0.9 mmol)at ambient temperature. After 10 min, crude amine 64 (125 mg, 0.31 mmol)in NMP (5 mL) was added in a dropwise fashion. After 16 h, the reactionmixture was diluted with diethyl ether and washed successively withNaHCO₃, 1 M HCl, water, and brine. The organic extract was dried overanhydrous Na₂SO₄, filtered and concentrated. The crude product waspurified by flash silica gel chromatography (EtOAc/hexane, 1:1 to 2:1)to afford 137 mg (76%) of 65 as a light yellow-colored solid. ¹H NMR(CDCl₃, 300 MHz) δ7.46 (d, J=7.8 Hz, 1H), 7.34 (d, J=7.8 Hz, 1H),7.19-7.12 (m, 3H), 7.05-6.99 (m, 2H), 6.70 (dd, J=2.4, 8.4 Hz, 1H),5.14-5.08 (m, 2H), 4.48-4.42 (m, 1H), 4.26-4.18 (m, 2H), 3.93 (app t,J=9.3 Hz, 1H), 3.84-3.76 (m, 1H), 3.47-3.31 (m, 2H), 2.84-2.64 (m, 4H),2.15-2.08 (m, 1H), 1.97-1.93 (m, 1H), 1.71-1.52 (m, 3H), 1.48 (s, 9H),1.38 (d, J=7.2 Hz, 3H), 1.18-1.12 (m, 1H), 0.64-0.56 (m, 1H) ppm.

G. Tyrosine-Derived Cyclic Ether (66)

To a solution of 65 (137 mg, 0.23 mmol) in DCM (10 mL) was added TFA (2mL) at ambient temperature. After 1.5 h, the solvent was removed underreduced pressure. The residue was dissolved in EtOAc and washed withaqueous NaHCO₃, brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was purified by reverse-phase HPLC (C18,10-100% ACN/water containing 0.1% HOAc) to afford 90 mg (82%) of 66 as awhite solid. ¹H NMR (DMSO, 300 MHz) 88.23 (d, J=6.9 Hz, 1H), 7.46-7.39(m, 3H), 7.23-7.12 (m, 2H), 7.01-6.99 (m, 2H), 6.85-6.82 (m, 1H), 4.86(m, 1H), 4.27 (m, 1H), 4.16 (m, 1H), 3.98-3.84 (m, 1H), 3.44-3.33 (m,1H), 3.16-3.10 (m, 1H), 2.76-2.67 (m, 3H), 1.99 (m, 1H), 1.57 (app t,J=5.7 Hz, 2H), 1.45-1.39 (m, 1H), 1.16 (d, J=6.3 Hz, 3H), 1.02-0.97 (m,1H), 0.58-0.49 (m, 1H) ppm. Mass spectrum: m/z 476.2 [M+H]⁺.

TABLE 13 R_(11, 12), MS K_(D) Entry A₁ R_(1b) Y Z X₂ R₁₄₋₁₇ (M + H)⁺Range 13-93 H Me Y linked H O R₁₁ 476.2 B with linked R₁₁ with Y

Example 14

This example illustrated pyrrolidine derivatives of Table 14

The Preparation of2S-Amino-N-{1S-[2S-(1H-indol-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-propionamide(76) A.trans-2S-(3-Methanesulfonyloxy-propenyl)-pyrrolidine-1-carboxylic acidtert-butyl ester (67)

To a solution of 22 (2 g, 8.8 mmol) in DCM (10 mL) was addedtriethylamine (2.5 mL, 17.6 mmol). The solution was cooled in ice bathand methanesulfonyl chloride (0.74 mL, 9.68 mmol) was added dropwise andstirred at room temperature for 30 min. Water (10 mL) was added and theproduct was extracted with DCM (3×50 mL). The organic layers werecombined and washed with 5 mL 1N HCl, water (10 mL), and brine, anddried over anhydrous Na₂SO₄. Solvent evaporated under reduced pressureto obtained 9.5 g of 67 which was used without purification. ¹H NMR(CDCl₃, 300 MHz): δ 4.4-4.0 (m, 2H), 3.42-3.21 (m, 3H), 3.0 (s, 3H),2-1.6 (m, 4H), 1.42 (s, 9H) ppm.

B.trans-2S-{3-[Acetyl-(2-iodo-phenyl)-amino]-propenyl}-pyrrolidine-1-carboxylicacid tert-butyl ester (68)

A solution of o-iodoacetanilide (1.1 g, 4.21 mmol) in DMF (10 mL) wascooled in ice bath and NaH (60% dispersion in mineral oil, 0.24 g, 6.31mmol) was added in portions and stirred at room temperature for 10 min.Crude mesylate 67 (1.28 g, 4.21 mmol) in DMF (5 mL) was added dropwiseat room temperature and stirred for 30 min. Water (10 mL) was added andthe product was extracted with diethyl ether (3×50 mL). The combinedether extracts were washed with water (3×50 mL) and brine, dried overNa₂SO₄. Solvent evaporated under reduced pressure and purified by flashsilica gel chromatography (3:1 hexanes/ethyl acetate) to afford 1.17 gof 68 as a white solid. ¹H NMR (CDCl₃, 300 MHz): δ 7.85 (d, J=9.9 Hz,1H), 7.4-7.0 (m, 3H), 5.6 (m, 1H), 5.28 (m, 1H), 4.86 (m, 1H), 3.3 (m,3H), 2.0-1.6 (m, 4H), 1.4 (s, 9H) ppm.

C. 2S-(1-Acetyl-1H-indol-3-ylmethyl)-pyrrolidine-1-carboxylic acidtert-butyl ester (69)

To a solution of 68 (1.1 g, 2.33 mmol) in DMF (15 mL) was added K₂CO₃(0.41 g, 3.02 mmol), NaHCO₂ (0.16 g, 2.44 mmol) followed bytetrabutylammonium chloride (0.64 g, 2.33 mmol) and Pd(OAc)₂ (0.02 g,0.07 mmol) and the reaction flask was immersed in a pre-heated oil bath(100° C.). After 40 min., water (10 mL) was added and the product wasextracted with diethyl ether (3×50 mL). The diethyl ether extracts werewashed with water (3×50 mL), brine and dried over Na₂SO₄. Solvent wasevaporated under reduced pressure and purified by flash silica gelchromatography (3:1 hexanes/ethyl acetate) to afford 0.37 g of 69 aswhite solid. ¹H NMR (CDCl₃, 300 MHz): δ 8.4 (s, 1H), 7.78-7.6 (dd,J=10.7, 10.7 Hz, 1H), 4.2-4.0 (m, 1H), 3.45-3.0 (m, 3H), 2.7-2.6 (m,1H), 2.6 (s, 3H), 1.9-1.65 (m, 4H), 1.5 (s, 9H) ppm.

D. 1-(3-Pyrrolidin-2S-ylmethyl-indol-1-yl)-ethanone (70)

To a solution of 69 (0.37 g, 1.08 mmol) in DCM (20 mL) was added TFA (4mL) and stirred at room temperature for 30 min. Aqueous NaHCO₃ (5 mL)was added and the reaction mixture was concentrated under reducedpressure. The product was extracted with DCM (3×50 mL) and the organicextract was washed with aqueous NaHCO₃, water and brine, and dried overanhydrous Na₂SO₄. Solvent was removed under reduced pressure andpurified by silica gel column chromatography (10:1 DCM/MeOH) to afford0.26 g of 70 as white solid. ¹H NMR (CDCl₃, 300 MHz): δ 8.6 (d, J=11 Hz,1h), 7.5 (d, J=11 Hz, 1H), 7.4-7.2 (m, 3H), 3.8-3.6 (m, 1H), 3.2-2.9 (m,4H), 2.8 (s, 3h), 2.2-1.6 (m, 4H) ppm.

E.{1S-[2S-(1-Acetyl-1H-indol-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-carbamicacid tert-butyl ester (71)

To a solution of Boc-L-valine (0.25 g, 1.18 mmol) in NMP (5 mL), wasadded HATU (0.44 mg, 1.18 mmol) followed by NMM (0.3 mL, 2.67 mmol).After 5 min., added 70 (0.25 g, 0.11 mmol) and stirred at roomtemperature for 30 min. Ethyl acetate (20 mL) was added and the organicsolution was washed with aqueous NaHCO₃ (10 mL), 1 N HCl (10 mL), water,and brine, dried over Na₂SO₄. Solvent was removed under reduced pressureand the product was purified by flash silica gel chromatography toafford 0.36 mg of 71 as a white solid. ¹H NMR (CDCl₃, 300 MHz): δ 8.4(m, 1H), 7.88 (d, J=11 Hz, 1H), 7.4-7.2 (m, 3H), 4.4-4.2 (m, 1H),3.8-3.4 (m, 3H), 3.38 (d, J=11 Hz, 1H), 2.65 (d, J=11 Hz, 1H), 2.6 (s,3H), 2.5-2.4 (m, 1H), 2.1-1.7 (m, 4H), 1.4 (s, 9H), 1.05 (d, J=5.5 Hz,3H), 0.95 (d, J=5.5 Hz, 3H) ppm.

F.1-[2S-(1-Acetyl-1H-indol-3-ylmethyl)-pyrrolidin-1-yl]-2S-amino-3-methyl-butan-1-one(72)

To a solution of 71 (0.36 g, 0.81 mmol) in DCM (20 mL) was added TFA (4mL) and the reaction mixture was stirred at room temperature for 30 min.Aqueous NaHCO₃ was added to the reaction mixture and TFA and DCM wereremoved under reduced pressure. The product was extracted with DCM (3×50mL) and the combined DCM extracts were washed with water and brine anddried over anhydrous Na₂SO₄. Solvent was removed under reduced pressureand the product was purified by flash silica gel chromatography (10:1DCM/MeOH) to afford 0.27 g of 72 as white solid. ¹H NMR (CDCl₃, 300MHz): δ 8.4 (m, 1H), 7.88 (d, J=11 Hz, 1H), 7.4-7.2 (m, 3H), 4.4-4.2 (m,1H), 3.8-3.4 (m, 3H), 3.38 (d, J=11 Hz, 1H), 2.65 (d, J=11 Hz, 1H), 2.6(s, 3H), 2.5-2.4 (m, 1H), 2.1-1.7 (m, 4H), 1.05 (d, J=5.5 Hz, 3H), 0.95(d, J=5.5 Hz, 3H) ppm.

G.(1S-{1S-[2S-(1-Acetyl-1H-indol-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propylcarbamoyl}-ethyl)-carbamicacid tert-butyl ester (73)

To a solution of Boc-L-alanine (0.13 g, 0.38 mmol) in NMP (3 mL) wasadded HATU (0.16 mg, 0.41 mmol) followed by NMM (0.1 mL, 0.95 mmol).After 5 min., 72 (0.12 g, 0.34 mmol) was added and the reaction mixturewas stirred at room temperature for 30 min. Ethyl acetate (20 mL) wasadded to the reaction mixture and the organic solution was washed withaqueous NaHCO₃ (10 mL), 1 N HCl (10 mL), water, and brine, dried overNa₂SO₄. Solvent was removed under reduced pressure and the product waspurified by flash silica gel chromatography to afford 0.15 mg of 73 aswhite solid. ¹H NMR (CDCl₃, 300 MHz): δ 8.4 (m, 1H), 7.88 (d, J=10.9 Hz,1H), 7.4-7.2 (m, 3H), 7.0-6.8 (m 1H), 4.6 (m, 1H), 4.5-4.4 (m, 1H), 4.2(m, 1H), 3.8-3.6 (m, 2H), 3.38 (d, J=10.9 Hz, 1H), 2.65 (d, J=10.9 Hz,1H), 2.6 (s, 3H), 2.5-2.4 (m, 1H), 2.1-1.7 (m, 4H), 1.45 (s, 9H), 1.4(d, J=10.9 Hz, 3H), 1.05 (d, J=5.5 Hz, 3H), 0.95 (d, J=5.5 Hz, 3H) ppm.

H.N-{1S-[2S-(1-Acetyl-1H-indol-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-2S-amino-propionamide(74)

To a solution of 73 (0.15 g, 0.29 mmol) in DCM (20 mL) was added TFA (4mL) and the reaction mixture was stirred at room temperature for 30 min.Aqueous NaHCO₃ was added to the reaction mixture and TFA and DCM wereremoved under reduced pressure. The product was extracted with DCM (3×50mL) and the DCM extracts were washed with water, brine, and dried overanhydrous Na₂SO₄. Solvent was removed under reduced pressure and theproduct was purified by flash silica gel chromatography (10:1 DCM/MeOH)to afford 0.12 g of 74 as a white solid. ¹H NMR (CDCl₃, 300 MHz): δ 8.4(m, 1H), 7.88 (d, J=10.9 Hz, 1H), 7.85 (d, J=10.9 Hz, 1H), 7.4-7.2 (m,3H), 4.6 (m, 1H), 4.5-4.4 (m, 1H), 3.8 (m, 1H), 3.75-3.4 (m, 2H), 3.38(d, J=10.9 Hz, 1H), 2.65 (d, J=10.9 Hz, 1H), 2.6 (s, 3H), 2.5-2.4 (m,1H), 2.1-1.7 (m, 4H), 1.4 (d, J=10.9 Hz, 3H), 1.05 (d, J=5.5 Hz, 3H),0.95 (d, J=5.5 Hz, 3H) ppm.

2S-Amino-N-{1S-[2S-(1H-indol-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-propionamide(75)

To a solution of 74 (0.12 g, 0.291 mmol) in dry MeOH (3 mL) was added 3mL of 10% NaOH/MeOH. After 15 min., water (2 mL) was added to thereaction mixture and the solvent was removed under reduced pressure. Theproduct was extracted with ethyl acetate (3×50 mL) and the organicextracts were washed with water, brine, and dried over Na₂SO₄. Solventwas removed under reduced pressure and the product was purified by flashsilica gel chromatography (10:1 DCM/MeOH) to afford 0.06 g of 75 aswhite solid. ¹H NMR (CDCl₃, 300 MHz): δ 8.4 (m, 1H), 7.88 (d, J=10.9 Hz,1H), 7.85 (d, J=10.9 Hz, 1H), 7.4-7.2 (m, 3H), 4.6 (m, 1H), 4.5-4.4 (m,1H), 3.8 (m, 1H), 3.75-3.4 (m, 2H), 3.38 (d, J=10.9 Hz, 1H), 2.65 (d,J=10.9 Hz, 1H), 2.5-2.4 (m, 1H), 2.1-1.7 (m, 4H), 1.4 (d, J=10.9 Hz,3H), 1.05 (d, J=5.5 Hz, 3H), 0.95 (d, J=5.5 Hz, 3H) ppm.

TABLE 14 MS K_(D) Entry A₁ R_(1b) Y Z R₁₂ R₁₄₋₁₇ R₁₁ (M + H)⁺ Range14-94 H Me iPr H Ac H H 413.2 A 14-95 H H iPr H Ac H H 399.3 D 14-96 MeMe tBu H Ac 6-F H 459.2 B (R₁₆) 14-97 Me Me cHex H Ac 6-F H 485.3 B(R₁₆) 14-98 H Me iPr H Ac 7-Me H 426.8 A (R₁₇) 14-99 H Me iPr H H H H371.3 B 14-100 H Me iPr H H 7-Me H 385.5 A (R₁₇) 14-101 Boc Me iPr H BocH H 571.3 D 14-102 H Me iPr H MeC≡CCH₂ H H 423.3 B 14-103 H Me iPr HHC≡CCH₂ H H 409.3 A 14-104 Me Me cHex H H 6-F H B (R₁₆) 14-105 H H tBu HAc 6-F H D

Example 15

This example illustrated pyrrolidine derivatives of Table 15.

The Preparation of2S-Amino-N-(2-methyl-1S-{2S-[1-(toluene-4-sulfonyl)-1H-indol-3-ylmethyl]-pyrrolidine-1-carbonyl}-propyl)-propionamide(78) A.(1S-{1S-[2S-(1H-Indol-3-ylmethyl)-pyrrolidine-1-carbonyl]-2-methyl-propylcarbamoyl}-ethyl)-carbamicacid tert-butyl ester (76)

To a solution of 73 (0.70 g, 1.36 mmol) in dry MeOH (10 mL) was added 5mL of 10% NaOH/MeOH and stirred for 15 min. Water (5 mL) was added tothe reaction mixture and the solvent removed under reduced pressure. Theproduct was extracted with ethyl acetate (3×50 mL) and the organicextracts were washed with water, brine, and dried over Na₂SO₄. Solventwas removed under reduced pressure and the product was purified by flashsilica gel chromatography (10:1 DCM/MeOH) to afford 0.53 g of 76 as awhite solid. ¹H NMR (CDCl₃, 300 MHz): δ 8.0 (s, 1H), 7.9 (d, J=9.9 Hz,1H), 7.38 (d, J=9.9 Hz, 1H), 7.3-7.1 (m, 3H), 6.8 (m, 1H), 4.62 (m 1H),4.5-4.4 (m 1H), 4.4-4.0 (m, 2H), 3.7-3.5 (m, 2H), 3.4 (m, 1H), 2.5 (m,1H), 2.2-1.8 (m, 4H), 1.48 (s, 9H), 1.35 (d, J=9.9 Hz, 3H), 1.05 (d,J=5.5 Hz, 3H), 0.95 (d, J=5.5 Hz, 3H) ppm.

B.[1S-(2-Methyl-1S-{2S-[1-(toluene-4-sulfonyl)-1H-indol-3-ylmethyl]-pyrrolidine-1-carbonyl}-propylcarbamoyl)-ethyl]-carbamicacid tert-butyl ester (77)

To a solution of 76 (0.05 g, 0.11 mmol) in DCM (1 mL) was added NaOH(0.01 g, 0.13 mmol) and stirred at room temperature for 30 min. Tosylchloride (0.03 g, 0.16 mmol) was added and the reaction mixture washeated to 35° C. for 1 h. Water (5 mL) was added to the reaction mixtureand the product was extracted with DCM (3×30 mL). The DCM extracts werewashed with water, brine, and dried over Na₂SO₄. Solvent was removedunder reduced pressure and the product was purified by flash silica gelchromatography (3:1 hexane/ethyl acetate) to afford 61 mg of 77 as awhite solid. ¹H NMR (CDCl₃, 300 MI-1z): δ 7.95 (d, J=9.9 Hz, 1H), 7.82(d, J=9.9 Hz, 1H), 7.7 (d, J=9.9 Hz, 2H), 7.4-7.2 (m, 5H), 6.8 (m, 1H),4.6 (m, 1H), 4.4 (m, 1H), 4.3-4.18 (m, 2H), 3.8-3.5 (m, 2H), 3.3 (m,1H), 2.45 (m, 1H), 2.35 (s, 3H), 2.2-1.8 (m, 4H), 1.43 (s, 9H), 1.38 (d,J=9.9 Hz, 3H), 1.05 (d, J=5.5 Hz, 3H), 0.95 (d, J=5.5 Hz, 3H) ppm.

C.2S-Amino-N-(2-methyl-1S-{2S-[1-(toluene-4-sulfonyl)-1H-indol-3-ylmethyl]-pyrrolidine-1-carbonyl}-propyl)-propionamide(78)

To a solution of 77 (0.06 g, 0.096 mmol) in DCM (10 mL) was added TFA (2mL) and the reaction mixture was stirred at room temperature for 30 min.Aqueous NaHCO₃ was added to reaction mixture and TFA and DCM wereremoved under reduced pressure. The product was extracted with DCM (3×25mL) and the DCM extracts were washed with water, brine, and dried overanhydrous Na₂SO₄. Solvent was removed under reduced pressure and theproduct was purified by flash silica gel chromatography (10:1 DCM/MeOH)to afford 0.04 g of 78 as a white solid. ¹H NMR (CDCl₃, 300 MHz): δ 7.95(d, J=9.9 Hz, 1H), 7.82 (d, J=9.9 Hz, 1H), 7.7 (d, J=9.9 Hz, 2H),7.4-7.2 (m, 5H), 6.8 (m, 1H), 4.6 (m, 1H), 4.4 (m, 1H), 4.3-4.18 (m,2H), 3.8-3.5 (m, 2H), 3.3 (m, 1H), 2.45 (m, 1H), 2.35 (s, 3H), 2.2-1.8(m, 4H), 1.38 (d, J=9.9 Hz, 3H), 1.05 (d, J=5.5 Hz, 3H), 0.95 (d, J=5.5Hz, 3H) ppm.

TABLE 15 MS K_(D) Entry A₁ R_(1b) Y Z R₁₂ R₁₄₋₁₇ R₁₁ (M + H)⁺ Range15-104 H Me iPr H MeC≡CCH₂ H H 423.3 B 15-105 H Me iPr H HC≡CCH₂ H H409.3 A 15-106 Me Me iPr H H 7-BnO H 491.3 C (R₁₇) 15-107 H Me iPr HTosyl H H 525.3 A 15-108 Me Me tBu H H 6-F 2- 499.5 A (R₁₆) thiophenyl

Example 16

This example illustrated pyrrolidine derivatives of Table 16.

The Preparation ofN-{1-Cyclohexyl-2-[2-(6-fluoro-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-1H-indol-3-ylmethyl)-pyrrolidin-1-yl]-2-oxo-ethyl}-2-methylamino-propionamide(85) A.2-(6-Fluoro-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-1H-indole-3-ylmethyl)-2S-pyrrolidine-1-carboxylicacid tert-butyl ester (80)

To a suspension of NaH (72 mg, 1.8 mmol, 60% mineral oil suspension) inDMF (1 mL) was added 79 (180 mg, 0.60 mmol) in DMF (1 mL) at roomtemperature. The mixture was stirred for 1 h at room temperature,followed by addition of tri(ethylene glycol)monomethyl ether (182 mg,0.57 mmol) in DMF (1 mL). The resulting mixture was stirred at roomtemperature overnight, and quenched by addition of NH₄Cl aqueoussolution at 0° C. The crude product was extracted with EtOAc. Theorganic phase was washed with brine and dried over Na₂SO₄. After removalof the solvent, the residue was purified by flash silica gelchromatography (50% EtOAc in hexanes) to afford 250 mg of 80 (93%). [TLC(60% EtOAc/hexane): R_(f)(80)=0.22)]. ¹H NMR (CDCl₃, 300 MHz) δ7.65-7.76 (1H), 7.33 (m, 1H), 7.20 (m, 1H), 7.09 (m, 1H), 6.95 (s, 1H),4.27 (t, J=8.4 Hz, 2H), 3.79 (t, J=5.7 Hz, 2H), 3.40-3.70 (9H), 3.37 (s,3H), 3.10-3.40 (2H), 2.65 (m, 1H), 1.75 (brs, 4H), 1.55 (s, 9H) ppm.

B.6-Fluoro-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-3-pyrrolidin-2-ylmethyl-1H-indole(81)

A solution of 80 (250 mg, 0.56 mmol) in DCM (5 mL) was treated with TFA(1 mL) at room temperature. After 2 h, the reaction mixture wasconcentrated, diluted with EtOAc, washed with 1N aqueous NaOH and brine,dried over Na₂SO₄, filtered and concentrated to afford 190 mg (98%) of81 as a light yellow oil. The product was used without furtherpurification.

C.{1-Cyclohexyl-2-[2-(6-fluoro-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-1H-indol-3-ylmethyl)-pyrrolidin-1-yl]-2-oxo-ethyl}-carbamicacid tert-butyl ester (82)

To a solution of Boc-L-cyclohexylglycine (172 mg, 0.67 mmol) in NMP (2mL) was added HATU (255 mg, 0.67 mmol) followed by NMM (0.1 mL, 0.95mmol). After 5 min, 81 (190 mg, 0.55 mmol) in DCM (1 mL) was added andthe reaction mixture was stirred at room temperature overnight. Ethylacetate (20 mL) was added to the reaction mixture and the organicsolution was washed with water, 1 N HCl (5 mL), aqueous NaHCO₃ (10 mL),and brine, dried over Na₂SO₄. Solvent was removed under reduced pressureand the product was purified by flash silica gel chromatography toafford 280 mg (85%) of 82 as a wax. [TLC (60% EtOAc/hexane):R_(f)(82)=0.18)]. ¹H NMR (CDCl₃, 300 MHz) δ 7.83 (m, 1H), 7.32 (m, 1H),7.20 (m, 1H), 7.09 (m, 1H), 6.98 (s, 1H), 5.32 (m, 1H), 4.0-4.6 (5H),3.79 (t, J=5.7 Hz, 2H), 3.40-3.70 (9H), 3.37 (s, 3H), 2.65 (m, 1H),1.6-2.0 (10H), 1.46 (s, 9H), 1.0-1.4 (6H) ppm.

D.2-Amino-2-cyclohexyl-1-[2-(6-fluoro-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-1H-indol-3-ylmethyl)-pyrrolidin-1-yl]-ethanone(83)

A solution of 82 (250 mg, 0.43 mmol) in DCM (5 mL) was treated with TFA(1 mL) at room temperature. After 2 h, the reaction mixture wasconcentrated, diluted with EtOAc, washed with 1N aqueous NaOH and brine,dried over Na₂SO₄, filtered and concentrated to afford 210 mg (100%) of83 as a light yellow wax. The product was used without furtherpurification.

E.(1-{1-Cyclohexyl-2-[2-(6-fluoro-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-1H-indol-3-ylmethyl)-pyrrolidin-1-yl]-2-oxo-ethylcarbamoyl}-ethyl)-methyl-carbamicacid tert-butyl ester (84)

To a solution of Boc-L-N-methylanaline (91 mg, 0.48 mmol) in NMP (2 mL)was added HATU (183 mg, 0.48 mmol) followed by NMM (0.1 mL, 0.95 mmol).After 5 min., 83 (210 mg, 0.43 mmol) in DCM (1 mL) was added and thereaction mixture was stirred at room temperature overnight. Ethylacetate (20 mL) was added to the reaction mixture and the organicsolution was washed with water, 1 N HCl (5 mL), aqueous NaHCO₃ (10 mL),and brine, dried over Na₂SO₄. Solvent was removed under reduced pressureand the product was purified by flash silica gel chromatography toafford 188 mg (65%) of 84 as a wax. [TLC (80% EtOAc/hexane):R_(f)(84)=0.20)]. ¹H NMR (CDCl₃, 300 MHz) δ 7.80 (m, 1H), 7.35 (m, 1H),7.19 (m, 1H), 7.10 (m, 1H), 6.95 (s, 1H), 4.62 (m, 1H), 4.46 (m, 1H),4.20 (t, J=6.7 Hz, 2H), 3.77 (, t, J=5.4 Hz, 2H), 3.2-3.7 (5H), 3.36 (s,3H), 2.48 (d, J=7.5 Hz, 3H) (t, J=5.7 Hz, 2H), 3.40-3.70 (9H), 3.37 (s,3H), 2.65 (m, 1H), 2.57 (m, 1H), 1.6-2.0 (8H), 1.51 (S, 9H), 1.38 (d,J=6.6 Hz, 3H), 1.0-1.4 (4H) ppm.

F.2-(6-Fluoro-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-1H-indole-3-ylmethyl)-2S-pyrrolidine-1-carboxylicacid tert-butyl ester (85)

A solution of 84 (188 mg, 0.28 mmol) in DCM (5 mL) was treated with TFA(1 mL) at room temperature. After 2 h, the reaction mixture wasconcentrated, diluted with EtOAc, washed with 1N aqueous NaOH and brine,dried over Na₂SO₄, filtered and concentrated. The residue was purifiedby HPLC to afford 110 mg (65%) acetate salt of 85 as a white solid. [TLC(20% MeOH in EtOAc): R_(f)(85)=0.20)]. ¹H NMR (CDCl₃, 300 MHz) δ 7.79(br d, 2H), 7.34 (d, J=7.2 Hz, 1H), 7.21 (m, 1H), 7.10 (m, 1H), 6.96 (d,J=9.0 Hz, 1H), 6.23 (br s, 2H), 4.61 (m, 1H), 4.44 (m, 1H), 4.24 (t,J=5.4 Hz, 2H), 3.78 (t, J=5.4 Hz, 2H), 3.4-3.8 (12H), 3.36 (s, 3H), 2.60(m, 1H), 2.48 (d, J=7.5 Hz, 3H), 1.6-2.0 (8H), 1.39 (d, J=6.6 Hz, 3H),1.0-1.4 (4H) ppm. Mass spectrum, m/z 571.3 [M+H]⁺.

TABLE 16 MS K_(D) Entry A₁ R_(1b) Y Z R₁₂ R₁₄₋₁₇ R₁₁ (M + H)⁺ Range16-108 Me Me tBu H MeO(CH₂CH₂O)₂CH₂CH₂ 6-F H 563.4 B (R₁₆) 16-109 Me MecHex H MeO(CH₂CH₂O)₂CH₂CH₂ 6-F H 589.4 B (R₁₆) 16-110 Me Et cHex HMeO(CH₂CH₂O)₂CH₂CH₂ H H 585.5 B 16-111 Me Me cHex H MeO(CH₂CH₂O)₂CH₂CH₂H H 571.5 B 16-112 Me Me tBu H HO(CH₂CH₂O)₂CH₂CH₂ 6-F Br 627.3 A (R₁₆)16-113 Me Me tBu H MeO(CH₂CH₂O)₂CH₂CH₂ 6-F Br 641.3 A (R₁₆) 16-114 Me MetBu H MeO(CH₂CH₂O)₂CH₂CH₂ 6-F 2- 645.4 A (R₁₆) thiophenyl

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontain within this specification.

What is claimed is:
 1. A compound of formula (5)

or a pharmaceutically acceptable salt thereof where A₁ is H, or methyl;R_(1a) is H; R_(1b) is methyl or ethyl; Y is an alkyl, an alkynyl, acycloalkyl of 3 to 7 carbon atoms, a heteroalkynyl, or optionallysubstituted versions of these groups, or Y together with R₁₀ forms acarbocyclic ring, or a heterocyclic ring containing 1 to 5 heteroatoms,where Y is linked to R₁₀; Z_(1a) and Z_(1b) are independently an H,hydroxy, alkoxy, or aryloxy; M is an optionally-substituted alkylene of1 to 5 carbon atoms; G is a bond, —O— or —NH— and R₁₀ is heteroaryl. 2.A pharmaceutical composition comprising a compound of claim 1, or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.
 3. The compound of claim 1 or a pharmaceuticallyacceptable salt thereof wherein R10 is a substituted heteroaryl.
 4. Thecompound of claim 1 wherein the heteroaryl is selected from the groupconsisting of benzofurans, benzo[b]thiophene 1-oxide, indoles,2-thienyls, 3-thienyls, thiophenyls, thiazoyls, pyrazines, andpyridines.
 5. The compound of claim 3 wherein the substituted heteroarylis selected from the group consisting of benzofurans, benzo[b]thiophene1-oxide, indoles, 2-thienyls, 3-thienyls, thiophenyls, thiazoyls,pyrazines, and pyridines.
 6. The compound of claim 5 wherein thesubstituted heteroaryl is a substituted thiazoyl.
 7. A pharmaceuticalcomposition comprising a compound of claim 6, or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.
 8. Amethod of treating cancerous cells comprising administering to the cellsan TAP binding compound of claim
 6. 9. A method of treating cellscomprising: administering to cells that have a proliferation disorder,wherein the disorder is selected from the group consisting of a canceror an autoimmune disorder, an amount of the TAP binding compound ofclaim 6 or a pharmaceutically acceptable salt thereof that reduces thecellular proliferation disorder in the sample of cells.
 10. A method oftreating cells comprising: administering to cells that have aproliferation disorder, wherein the disorder is selected from the groupconsisting of a cancer or an autoimmune disorder, an amount of the TAPbinding compound of claim 6 or a pharmaceutically acceptable saltthereof that reduces the cellular proliferation disorder in the sampleof cells.
 11. A method of treating a cellular proliferative disorder ina patient, wherein the proliferative disorder is selected from the groupconsisting of a cancer or an autoimmune disorder, the method comprisingadministering to the patient an TAP binding compound of claim 1 in anamount that ameliorates the cellular proliferative disorder.
 12. Themethod of claim 11 wherein the TAP binding compound is an TAP bindingcompound of claim
 5. 13. The method of claim 11 wherein the TAP bindingcompound is an TAP binding compound of claim
 6. 14. The compound ofclaim 1 or a pharmaceutically acceptable salt wherein Y is an alkyl or acycloalkyl of 3 to 7 carbon atoms; Z_(1a) and Z_(1b) are independently—H, hydroxy or alkoxy; and R₁₀ is heteroaryl.
 15. The compound of claim14 or a pharmaceutically acceptable salt wherein M is substitutedmethylene; G is —NH—; and R₁₀ is substituted thiazoyl.